TECHNICAL FIELD OF THE INVENTION
[0001] The invention relates to the area of regulation of kinase activity. More particularly,
the invention relates to the regulation of a novel human kinase, Regulated in COPD
Kinase (RC Kinase). The invention discloses that the gene of RC Kinase is overexpressed
in COPD patients and is useful as a diagnostic marker and target for treatment. Methods
are disclosed for predicting, diagnosing and prognosing as well as preventing and
treating COPD with the use of RC Kinase. Methods are also disclosed for predicting,
diagnosing and prognosing as well as preventing and treating other ailments in which
RC Kinase is dysregulated or in which modulation or enhancement of the activity of
RC Kinase can modify disease progression. Modulation of RC Kinase activity can affect
disease status such as, COPD, asthma, cancer, Alzheimer's disease, inflammatory diseases,
and caldiovascular diseases.
BACKGROUND OF THE INVENTION
[0002] Intracellular signaling regulates a variety of important biological functions. One
common method used by cells to conduct signals is protein phosphorylation. In order
to transmit signals, activated enzymes called protein kinases attach phosphate groups
to downstream molecules in a signaling cascade and thereby, depending on the type
of molecule, regulate their enzymatic activity, their subcellular localization, their
interaction with other molecules, their shape, or their halflife. One important family
of protein kinases involved in this type of signal transmission is the mitogen-activated
protein kinases (MAPKs) (
Widmann, C. et al., Physiol Rev 79(1):143-80, 1999). Because MAPKs are themselves regulated by phosphorylation, they are often members
of complex phosphorelay systems within cells involving other kinases. For example
a MAPK can be phosphorylated by MAPK kinases (MKKs), which in turn can be phosphorylated
by MAPK kinase kinases (MKKKs). Such a phosphorelay system can serve to amplify a
signal, determine the specificity of a signal, and allow regulation at different points
in the signaling cascade. MAPKs, MKKs, and MKKKs have been found to play roles in
a large variety to cellular activities, including gene expression, mitosis, proliferation,
cell movement, metabolism, and programmed cell death. Because of the important functions
of protein kinase family enzymes such as the MAPKs, MKKs, and MKKKs, there is a need
in the art to identify new MAPK pathway kinases and methods of regulating these new
kinases for therapeutic effects.
[0003] WO 2002/33099 discloses a polynucleotide encoding a PKIN-6 kinase with decreased expression in
COPD, wherein the polynucleotide has a certain degree of sequence identity compared
to the polynucleotides of the present invention.
WO 2003/018786 discloses a polynucleotide encoding a serine-threonin kinase suitable for the treatment
of COPD, wherein the polynucleotide has no relevant degree of sequence identity compared
to the polynucleotides of the present invention.
SUMMARY OF THE INVENTION
[0004] It is an object of the invention to provide reagents and methods of regulating a
human RC Kinase. These and other objects of the invention are provided by one or more
of the embodiments described below.
[0005] One embodiment of the invention is an isolated polynucleotide encoding a RC Kinase
polypeptide, a serine/threonine kinase, with an increased expression in chronic obstructive
pulmonary disease patients selected from the group consisting of:
- a) a polynucleotide encoding a RC Kinose polypeptide comprising an amino acid sequence
selected frome the group consisting of:
amino acid sequences which are at least about 75% identical to
the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12; and
the amino acid sequnence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12.
- b) a polynucleotide comprising the sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6; and
- c) a polynucleotide comprising a nucleotide sequence which deviates from the polynucleotide
sequences specified in (a) to (b) due to the degeneration of the genetic code.
[0006] Another embodiment of the invention is a substantially purified RC Kinase polypeptide
encoded by a polynucleotide of the above.
[0007] The RC Kinase polypeptides of the present invention are suitable for novel preventive,
predictive, diagnostic, prognostic and therapeutic compositions and uses for COPD.
Since RC Kinase expression levels are increased in the disease state, its gene product
is a particularly useful target for treatment methods as well as diagnostic and clinical
monitoring methods.
[0008] Disclosed herein are also novel preventive, predictive, diagnostic, prognostic and
therapeutic compositions and uses for COPD based on derivatives, fragments, analogues
and homologues of the RC Kinase gene.
[0009] The present invention further relates to methods for detecting the dysregulation
of RC Kinase in COPD on the DNA and mRNA levels.
[0010] The present invention further relates to an
in vitro method for the prediction, diagnosis or prognosis of respiratory diseases or COPD
by the detection of RC Kinase gene or RC Kinase genomic nucleic acid sequence which
is altered in COPD.
[0011] In one embodiment the expression of the RC Kinase gene can be detected with arrays.
[0012] In a further embodiment, the expression of the gene can be detected with bead based
direct fluorescent readout techniques such as provided by Luminex corporation (
US 6,268,222).
[0013] The invention relates to a method of screening for agents which decrease the activity
of a RC Kinase polypeptide or a substantially purified RC Kinase polypeptide encoded
by a polynucleotide as described above, comprising the steps of: (a) contacting a
test compound with a polypeptide encoded by any polynucleotide as described above;
and (b) detecting binding of the test compound to the polypeptide, wherein a test
compound which binds to the polypeptide is identified as a potential therapeutic agent
for decreasing the activity of said polypeptide.
[0014] The invention also relates to a method of screening for agents which regulate the
activity of a RC Kinase polypeptide, comprising the steps of : (a) contacting a test
compound with a RC Kinase polypeptide encoded by any polynucleotide as described above;
and (b) detecting phosphorylation activity of the RC Kinase polypeptide, wherein a
test compound which increases the activity of the polypeptide is identified as a potential
therapeutic agent for increasing the activity of the RC Kinase polypeptide, and wherein
a test compound which decreases the activity of the polypeptide is identified as a
potential therapeutic agent for decreasing the activity of the RC Kinase polypeptide.
[0015] The invention also relates to a method of screening for agents which regulate the
activity of a RC Kinase, comprising the steps of: (a) contacting a test compound with
a RC Kinase polypeptide encoded by any polynucleotide as described above and MKK4;
and (b) detecting RC Kinase polypeptide phosphorylation of MKK4, wherein a test compound
which increases the RC Kinase polypeptide phosphorylation of MKK4 is identified as
a potential therapeutic agent for increasing the activity of the RC Kinase polypeptide,
and wherein a test compound which decreases the RC Kinase polypeptide phosphorylation
of MKK4 is identified as a potential therapeutic agent for decreasing the activity
of the RC Kinase polypeptide
[0016] The invention also relates to a method of screening for agents which decrease the
activity of a RC Kinase polypeptide, comprising contacting a test compound with any
polynucleotide as described above and detecting binding of the test compound to the
polynucleotide, wherein a test compound which binds to the polynucleotide is identified
as a potential therapeutic agent for decreasing the activity of RC Kinase polypeptide.
[0017] Further, the invention relates to a method of reducing the activity of RC Kinase,
comprising contacting a cell outside the human or animal body with a reagent which
specifically binds to any polynucleotide as described above or any RC Kinase polypeptide
as described above, wherein the reagent is a ribozyme, an antisense oligonucleotide
or an antibody, whereby the activity of RC Kinase is reduced.
[0018] The invention thus provides polypeptides if SEQ ID NO: 7, 8, 9, 10, 11, or 12 or
a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID
NO: 1, 2, 3, 4, 5, or 6 which can be used to identify compounds which may act, for
example, as regulators or modulators such as agonists and antagonists, partial agonists,
inverse agonists, activators, co-activators and inhibitors of the polypeptide comprising
the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a
polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6. Accordingly,
the invention provides reagents and methods for regulating a polypeptide comprising
the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a
polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in
COPD. The regulation can be an up- or down regulation. Reagents that modulate the
expression, stability or amount of a polynucleotide comprising a polynucleotide of
SEQ ID NO: 1, 2, 3, 4, 5, or 6 or the activity of the polypeptide comprising the polypeptide
of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide
comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 can be a protein,
a peptide, a peptidomimetic, a nucleic acid, a nucleic acid analogue (e.g. peptide
nucleic acid, locked nucleic acid) or a small molecule. Methods that modulate the
expression, stability or amount of a polynucleotide comprising a polynucleotide of
SEQ ID NO: 1, 2, 3, 4, 5, or 6 or the activity of the polypeptide comprising the polypeptide
of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a polynucleotide
comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 can be gene replacement
therapies, antisense, ribozyme, RNA interference and triplex nucleic acid approaches.
[0019] Disclosed herein are antibodies which specifically bind to a full-length or partial
polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide
encoded by a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4,
5, or 6 or a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4,
5, or 6 for use in prediction, prevention, diagnosis, prognosis and treatment of COPD.
[0020] Disclosed herein is the use of a reagent which specifically binds to a polynucleotide
comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a polypeptide comprising
the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a polypeptide encoded by a
polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in
the preparation of a medicament for the treatment of COPD.
[0021] Disclosed herein is the use of a reagent that modulates the activity or stability
of a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12 or
a polypeptide encoded by a polynucleotide comprising the polynucleotide of SEQ ID
NO: 1, 2, 3, 4, 5, or 6 or the expression, amount or stability of a polynucleotide
comprising a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 in the preparation of
a medicament for the treatment of COPD.
[0022] Disclosed herein is a pharmaceutical composition which includes a reagent which specifically
binds to a polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4,
5, or 6 or a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11,
or 12 or a polypeptide encoded by a polynucleotide comprising the polynucleotide of
SEQ ID NO: 1, 2, 3, 4, 5, or 6, and a pharmaceutically acceptable carrier.
[0023] Disclosed herein is a pharmaceutical composition including a polynucleotide comprising
the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or encoding a polypeptide comprising
the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12.
[0024] Disclosed herein is a reagent which alters the level of expression in a cell of a
polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or
encoding a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or
12, or a sequence complementary thereto, is identified by providing a cell, treating
the cell with a test reagent, determining the level of expression in the cell of a
polynucleotide comprising the polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or
encoding a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or
12 or a sequence complementary thereto, and comparing the level of expression of the
polynucleotide in the treated cell with the level of expression of the polynucleotide
in an untreated cell, wherein a change in the level of expression of the polynucleotide
in the treated cell relative to the level of expression of the polynucleotide in the
untreated cell is indicative of an agent which alters the level of expression of the
polynucleotide in a cell.
[0025] Disclosed herein is a pharmaceutical composition comprising a reagent identified
by this method.
[0026] The RC Kinase polypeptides of the present invention are suitable for use in a pharmaceutical
composition which includes a polypeptide comprising the polypeptide of SEQ ID NO:
7, 8, 9, 10, 11, or 12 or which is encoded by a polynucleotide comprising the polynucleotide
of SEQ ID NO: 1, 2, 3, 4, 5, or 6.
[0027] The invention also relates to the use of a screening method, said method comprising
the steps of
- (a) contacting a test compound with a polypeptide comprising an amino acid sequence
which is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, and
- (b) detecting binding of said test compound to a polypeptide of (a),
for the identification of compounds useful in the treatment of COPD.
[0028] Further, the invention relates to the use of a screening method, said method comprising
the steps of
- (a) contacting a test compound with a polypeptide comprising an amino acid sequence
which is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, and
- (b) detecting an activity of a polypeptide of (a),
for the identification of compounds useful in the treatment of COPD.
[0029] Disclosed herein is a pharmaceutical composition comprising a polynucleotide including
a sequence which hybridizes under stringent conditions to a polynucleotide comprising
a polynucleotide of SEQ ID NO: 1, 2, 3, 4, 5, or 6 and encoding a polypeptide exhibiting
the same biological function as RC Kinase, or encoding a polypeptide of SEQ ID NO:
7, 8, 9, 10, 11, or 12. Pharmaceutical compositions disclosed herein may further include
fusion proteins comprising a polypeptide comprising a polynucleotide of SEQ ID NO:
1, 2, 3, 4, 5, or 6, or a fragment thereof, antibodies, or antibody fragments
BRIEF DESCRIPTION OF THE DRAWINGS
[0030]
- Fig. 1
- shows the top 20 upregulated genes in lung tissue from COPD patients as determined
by microarray analysis. The fold increase in expression of these genes compared with
the average expression in lung tissue from normal subjects is shown in the far right
column.
RC Kinase is shown to have an increased expression of 4.58 fold in COPD compared to
normal, and is ranked 18th among the approximately 10,000 genes represented on the microarray.
- Fig. 2
- shows the relative expression levels for human RC Kinase obtained from microarray
experiments using various normal and COPD lung samples.
- Fig. 3
- shows confirmation by quantitative RT-PCR of the relative expression levels of RC
Kinase in normal and COPD lung. The results, generated on the same set of tissues
as used in the microarray analysis, correlate well with the microarray experiment
results shown in figure 2.
- Fig. 4
- shows the quantitative RT-PCR expression profiles of human RC Kinase in various tissues.
- Fig. 5
- shows the quantitative RT-PCR expression profiles of human RC Kinase in various immune
related cells, primary lung cell lines, and immortalized cell lines.
- Fig. 6
- shows changes in the expression level of human RC Kinase in the HEK293 cell line in
response to treatment with various stimuli that cause cell growth or cellular stress.
RC Kinase expression is upregulated after stimulation with 95 mM KCl.
- Fig. 7
- shows changes in the expression level of human RC Kinase in the Jurkat and Daudi cell
lines in response to treatment with various stimuli that cause cell growth or cellular
stress. Both Jurkat and Daudi cells show upregulation of RC Kinase expression after
stimulation with 95 mM KCI, and Jurkat cells show upregulation of RC Kinase expression
after stimulation with 500 µM H2O2.
- Fig. 8
- shows the phosphorylating activity of human RC Kinase on various MAP Kinase Kinases
tested as substrates. RC Kinase was able to phosphorylate itself and MKK4, but showed
only minor activity against MKK6 and no detectable activity against MEK2. RC Kinase
was prepared by immunoprecipitation of lysates of RC Kinase transfectants, then added
to a mixture of substrate and [33P]-ATP. Phosphorylation activity was detected by autoradiography after incubation
and size separation on SDS-PAGE. As controls, immuno-precipitate from an empty vector-transfectant
and heat-inactivated immunoprecipitate from an RC Kinase transfectant were used.
- Fig. 9
- shows the ability of human RC Kinase to induce the activation of the transcription
factors AP-1 and NFκB. An RC Kinase expression vector or an empty vector was transfected
into HEK293 cells together with luciferase reporter gene constructs for the transcription
factors AP-1, NFAT, NFκB, and the TATA-like promoter. Luciferase activity (expressed
in relative light units (RLU) compared with the TATA-like promoter's background luciferase
production) was measured 48 hours after transfection.
- Fig. 10
- shows the enhanced production of Interleukin-8 in HEK293 cells transfected with an
RC Kinase expression construct. IL-8 protein levels in the culture medium were measured
by an enzyme-linked immunosorbent assay 48 hours after the transfection of either
an empty vector or an RC Kinase expression construct.
- Fig. 11 shows
- an illustration indicating the role of RC Kinase in a MAP kinase phosphorelay system.
RC Kinase is activated in response to receptor-mediated signaling, stress, mitogens,
or other stimuli, or its expression is upregulated in response to such stimuli. As
a result, MKK4 and possibly other substrates are phosphorylated, leading eventually
to the activation of the transcription factors NFκB and AP-1. The transcription factors
then contribute to the upregulation of expression of IL-8 and other inflammatory mediators,
contributing to inflammation and other COPD-related pathology.
DETAILED DESCRIPTION OF THE INVENTION
[0031] "RC Kinase" as used herein refers to the polypeptide of SEQ ID NO 7, 8, 9, 10, 11
or 12, as well as it derivatives, fragments, analogs, and homologues thereof, or the
polypeptides encoded by the polynucleotide of SEQ ID NO: 1 as well as derivatives,
fragments, analogs and homologues thereof.
[0032] SEQ ID NO: 1 shows variant 1 of the DNA-sequence encoding an RC Kinase polypeptide.
This variant is 3719 bp in length, with an open reading frame extending from bases
1-3679 of the sequence. SEQ ID NO: 2 shows variant 2 of the DNA-sequence encoding
an RC Kinase polypeptide. This variant is 3338 bp in length, with an open reading
frame extending from bases 1-3243 of the sequence. SEQ ID NO: 3 shows variant 3 of
the DNA-sequence encoding an RC Kinase polypeptide. This variant is 3510 bp in length,
with an open reading frame extending from bases 1-3415 of the sequence. SEQ ID NO:
4 shows variant 4 of the DNA-sequence encoding an RC Kinase polypeptide. This variant
is 4058 bp in length, with an open reading frame extending from bases 1-4018 of the
sequence. SEQ ID NO: 5 shows variant 5 of the DNA-sequence encoding an RC Kinase polypeptide.
This variant is 1460 bp in length, with an open reading frame extending from bases
1-1420 of the sequence. SEQ ID NO: 6 shows variant 6 of the DNA-sequence encoding
an RC Kinase polypeptide. This variant is 1604 bp in length, with an open reading
frame extending from bases 1-1564 of the sequence. SEQ ID NO: 7 shows the amino acid
sequence deduced from the DNA-sequence of SEQ ID NO: 1. SEQ ID NO: 8 shows the amino
acid sequence deduced from the DNA-sequence of SEQ ID NO: 2.
[0033] SEQ ID NO: 9 shows the amino acid sequence deduced from the DNA-sequence of SEQ ID
NO: 3. SEQ ID NO: 10 shows the amino acid sequence deduced from the DNA-sequence of
SEQ ID NO: 4. SEQ ID NO: 11 shows the amino acid sequence deduced from the DNA-sequence
of SEQ ID NO: 5. SEQ ID NO: 12 shows the amino acid sequence deduced from the DNA-sequence
of SEQ ID NO: 6.
[0034] Furthermore, the activity of a novel RC Kinase, particularly a human RC Kinase, is
a discovery of the present invention. Human RC Kinase contains a single S_TKc kinase
domain (Serine/- Threonine protein kinases, catalytic domain), beginning approximately
268 amino acid residues from the carboxy terminal of SEQ ID NO: 7, 8, 10 or 12 and
spanning approximately 256 residues. Two of the variants of Human RC Kinase, SEQ ID
NO: 9 and 11, are missing part of this kinase domain. The kinase domain of Human RC
Kinase is highly homologous to the kinase domains of other known kinase type enzymes.
Human RC Kinase as shown in SEQ ID NO: 7, 8, 10 or 12 is 44% identical and 67% similar
over 287 amino acids (kinase domain) to the slime mold
Dictyostelium discoideum protein identified by GenBank Accession No. AAC97114 and annotated as a "MEK kinase
alpha." Similarly, human RC Kinase as shown in SEQ ID NO: 7,8, 10, or 12 is 47% identical
and 67% similar over 276 amino acids (kinase domain) to the common tobacco
Nicotiana tabacum protein identified by GenBank Accession No. A48084 and annotated as a "STE11 protein
kinase homolog NPK1," and is 46% identical and 63% similar over 291 amino acids (kinase
domain) to the human protein identified by GenBank Accession No. NP_002392 and annotated
as a "MAP/ERK kinase kinase 3; MAPKKK3." gene containing these coding sequences is
located within the human chromosome 2 genomic contig identified with GenBank accession
no. NT_005058, and is divided into at least 11 exons spanning more than 61,000 bases
of the genome. If the 11 3'-most exons are labeled as exons C, D, E, F, G, H, I, J,
K, L, and M, respectively, reading in order along the gene from 5' to 3', then six
alternative splice variants are described by the invention as follows. SEQ ID NO:
1 describes a splice variant which uses all exons except exons E, G, and H. SEQ ID
NO: 2 describes a splice variant which uses all exons except a portion of exon L.
SEQ ID NO: 3 describes a splice variant which uses all exons except exons E and a
portion of L. SEQ ID NO: 4 describes a splice variant which uses all exons except
exon E. SEQ ID NO: 5 describes a splice variant which uses all exons except exons
E, J, and a portion of L. SEQ ID NO: 6 describes a splice variant which uses all exons
except exons E, and J.
[0035] A single exon containing most of the kinase catalytic domain has previously been
annotated as "hypothetical protein FLJ23074," a gene with protein product and function
unknown.
[0036] RCKinase has the ability to phosphorylate other RC Kinase polypeptides, MKK4, and
MKK6. Taken together with the fact of importance of MAPK signalling, the modification
of RC kinase activity can give chance of remedy against COPD, asthma, cancer, Alzheimer's
disease, inflammatory diseases, and caldiovascular diseases.
Polypeptides
[0037] RC Kinase polypeptides according to the invention comprise the amino acid sequence
shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a biologically active variant thereof,
as defined below. A RC Kinase polypeptide of the invention therefore can be a portion
of a RC Kinase molecule, a full-length RC Kinase molecule, or a fusion protein comprising
all or a portion of a RC Kinase molecule. The invention also relates to a substantially
purified RC Kinase polypeptide encoded by a polynucleotide as described above.
Biologically Active Variants
[0038] RC Kinase variants which are biologically active,
i.
e., retain a RC Kinase activity, also are RC Kinase polypeptides. Preferably, naturally
or non-naturally occurring RC Kinase variants have amino acid sequences which are
at least about 50, preferably about 70, 75, 90, 96, or 98% identical to an amino acid
sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12. Percent identity between a putative
RC Kinase variant and an amino acid sequence of SEQ ID NO: 7, 8, 9, 10, 11, or 12
is determined by conventional methods. See, for example,
Altschul et al., Bull. Math. Bio. 48:603 (1986), and
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores
using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62"
scoring matrix of Henikoff & Henikoff, 1992.
[0039] Those skilled in the art appreciate that there are many established algorithms available
to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson
& Lipman is a suitable protein alignment method for examining the level of identity
shared by an amino acid sequence disclosed herein and the amino acid sequence of a
putative variant. The FASTA algorithm is described by
Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444(1988), and by
Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared
by the query sequence (e.g., SEQ ID NO: 2) and a test sequence that have either the
highest density of identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions, insertions, or
deletions. The ten regions with the highest density of identities are then rescored
by comparing the similarity of all paired amino acids using an amino acid substitution
matrix, and the ends of the regions are "trimmed" to include only those residues that
contribute to the highest score. If there are several regions with scores greater
than the "cutoff" value (calculated by a predetermined formula based upon the length
of the sequence the ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate alignment with
gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned
using a modification of the Needleman-Wunsch- Sellers algorithm (
Needleman & Wunsch, J. Mol. Biol.48:444 (1970);
Sellers, SIAM J. Appl. Math.26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for
FASTA analysis are: ktup=1, gap opening penalty=10, gap extension penalty=1, and substitution
matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying
the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0040] FASTA can also be used to determine the sequence identity of nucleic acid molecules
using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value
can range between one to six, preferably from three to six, most preferably three,
with other parameters set as default..
[0041] Variations in percent identity can be due, for example, to amino acid substitutions,
insertions, or deletions. Amino acid substitutions are defined as one for one amino
acid replacements. They are conservative in nature when the substituted amino acid
has similar structural and/or chemical properties. Examples of conservative replacements
are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate,
or a threonine with a serine.
[0042] Amino acid insertions or deletions are changes to or within an amino acid sequence.
They typically fall in the range of about 1 to 5 amino acids. Guidance in determining
which amino acid residues can be substituted, inserted, or deleted without abolishing
biological or immunological activity can be found using computer programs well known
in the art, such as DNASTAR software. Whether an amino acid change results in a biologically
active RC Kinase polypeptide can readily be determined by assaying for fibronectin
binding or for RC Kinase activity, as is known in the art and described, for example,
in Example 2.
Fusion Proteins
[0043] Fusion proteins are useful for generating antibodies against RC Kinase amino acid
sequences and for use in various assay systems. For example, fusion proteins can be
used to identify proteins which interact with portions of a RC Kinase polypeptide,
including its active site. Methods such as protein affinity chromatography or library-based
assays for protein-protein interactions, such as the yeast two-hybrid or phage display
systems, can be used for this purpose. Such methods are well known in the art and
also can be used as drug screens.
[0044] A RC Kinase fusion protein comprises two protein segments fused together by means
of a peptide bond. Contiguous amino acids for use in a fusion protein can be selected
from the amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or from a
biologically active variant thereof, such as those described above. Preferably, a
fusion protein comprises a kinase domain and/or an ATP binding site of human RC Kinase.
The first protein segment also can comprise full-length RC Kinase.
[0045] The second protein segment can be a full-length protein or a protein fragment or
polypeptide. Proteins commonly used in fusion protein construction include β- galactosidase,
β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including
blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish
peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope
tags are used in fusion protein constructions, including histidine (His) tags, FLAG
tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)
tags. Other fusion constructions can include maltose binding protein (MBP), S-tag,
Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes
simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered
to contain a cleavage site located between the RC Kinase polypeptide-encoding sequence
and the heterologous protein sequence, so that the RC Kinase polypeptide can be cleaved
and purified away from the heterologous moiety.
[0046] A fusion protein can be synthesized chemically, as is known in the art. Preferably,
a fusion protein is produced by covalently linking two protein segments or by standard
procedures in the art of molecular biology. Recombinant DNA methods can be used to
prepare fusion proteins, for example, by making a DNA construct which comprises RC
Kinase coding sequences disclosed herein in proper reading frame with nucleotides
encoding the second protein segment and expressing the DNA construct in a host cell,
as is known in the art. Many kits for constructing fusion proteins are available from
companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH
(Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International
Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).
Identification of Species Homologs
[0047] Species homologs of human RC Kinase can be obtained using RC Kinase polynucleotides
(described below) to make suitable probes or primers to screening cDNA expression
libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which
encode homologs of RC Kinase, and expressing the cDNAs as is known in the art.
Polynucleotides
[0048] A RC Kinase polynucleotide can be single- or double-stranded and comprises a coding
sequence or the complement of a coding sequence for a RC Kinase polypeptide. A partial
coding sequence of a RC Kinase polynucleotide is shown in SEQ ID NO: 1, 2, 3, 4, 5,
or 6; coding sequences of RC Kinase also are contained within the genomic sequence
shown in SEQ ID NO: 3, from nucleotides 11885 to 12023 and from nucleotides 10564
to 10693.
[0049] Degenerate nucleotide sequences encoding human RC Kinase polypeptides, as well as
homologous nucleotide sequences which are at least about 50, preferably about 75,
90, 96, or 98% identical to the RC Kinase coding sequences nucleotide sequences shown
in SEQ ID NOS: 1 and 3 also are RC Kinase polynucleotides. Percent sequence identity
between the sequences of two polynucleotides is determined using computer programs
such as ALIGN which employ the FASTA algorithm, using an affine gap search with a
gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA)
molecules, species homologs, and variants of RC Kinase polynucleotides which encode
biologically active RC Kinase polypeptides also are RC Kinase polynucleotides.
[0050] The invention relates to an isolated polynucleotide encoding a RC Kinase polypeptide,
a serine/threonine kinase, with an increased expression in chronic obstructive pulmonary
disease patients selected from the group consisting of:
- (a) a polynucleotide encoding a RC Kinase polypeptide, which polypeptide comprises
an amino acid sequence selected from the group consisting of:
- (i) an amino acid sequences which are at least 75% identical to the amino acid sequence
shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12; and
- (ii) an amino acid sequence as depicted in SEQ ID NO: 7, 8, 9,10,11, or 12.
- (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4,
5, or 6; and
- (c) a polynucleotide comprising a nucleotide sequence which deviates from the polynucleotide
sequences specified in (a) to (b) due to the degeneration of the genetic code.
Identification of Variants and Homologs
[0051] Variants and homologs of the RC Kinase polynucleotides disclosed above also are RC
Kinase polynucleotides. Typically, homologous RC Kinase polynucleotide sequences can
be identified by hybridization of candidate polynucleotides to known RC Kinase polynucleotides
under stringent conditions, as is known in the art. For example, using the following
wash conditions-2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room
temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50°C once, 30 minutes;
then 2X SSC, room temperature twice, 10 minutes each--homologous sequences can be
identified which contain at most about 25-30% basepair mismatches. More preferably,
homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably
5-15% basepair mismatches.
[0052] Species homologs of the RC Kinase polynucleotides disclosed herein can be identified
by making suitable probes or primers and screening cDNA expression libraries from
other species, such as mice, monkeys, or yeast. Human variants of RC Kinase polynucleotides
can be identified, for example, by screening human cDNA expression libraries. It is
well known that the T
m of a double-stranded DNA decreases by 1-1.5°C with every 1% decrease in homology
(
Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of human RC Kinase polynucleotides or RC Kinase polynucleotides of other
species can therefore be identified, for example, by hybridizing a putative homologous
RC Kinase polynucleotide with a polynucleotide having a nucleotide sequence of SEQ
ID NO: 1, 2, 3, 4, 5, or 6 or an ephrin-like serine protease coding sequence of SEQ
ID NO: 3 to form a test hybrid. The melting temperature of the test hybrid is compared
with the melting temperature of a hybrid comprising RC Kinase polynucleotides having
perfectly complementary nucleotide sequences, and the number or percent of basepair
mismatches within the test hybrid is calculated.
[0054] Typically, for stringent hybridization conditions a combination of temperature and
salt concentration should be chosen that is approximately 12-20°C below the calculated
T
m of the hybrid under study. The T
m of a hybrid between a RC Kinase polynucleotide having a coding sequence disclosed
herein and a polynucleotide sequence which is at least about 50, preferably about
75, 90, 96, or 98% identical to that nucleotide sequence can be calculated, for example,
using the equation of
Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

where
l = the length of the hybrid in basepairs.
[0055] Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide,
4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include,
for example, 0.2X SSC at 65°C.
Preparation of Polynucleotides
[0056] A naturally occurring RC Kinase polynucleotide can be isolated free of other cellular
components such as membrane components, proteins, and lipids. Polynucleotides can
be made by a cell and isolated using standard nucleic acid purification techniques,
synthesized using an amplification technique, such as the polymerase chain reaction
(PCR), or synthesized using an automatic synthesizer. Methods for isolating polynucleotides
are routine and are known in the art. Any such technique for obtaining a polynucleotide
can be used to obtain isolated RC Kinase polynucleotides. For example, restriction
enzymes and probes can be used to isolate polynucleotide fragments which comprise
RC Kinase nucleotide sequences. Isolated polynucleotides are in preparations which
are free or at least 70, 80, or 90% free of other molecules.
[0057] RC Kinase cDNA molecules can be made with standard molecular biology techniques,
using RC Kinase mRNA as a template. RC Kinase cDNA molecules can thereafter be replicated
using molecular biology techniques known in the art and disclosed in manuals such
as Sambrook
et al. (1989). An amplification technique, such as PCR, can be used to obtain additional
copies of RC Kinase polynucleotides, using either human genomic DNA or cDNA as a template.
[0058] Alternatively, synthetic chemistry techniques can be used to synthesize RC Kinase
polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences
to be synthesized which will encode a RC Kinase polypeptide having, for example, the
amino acid sequence shown in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a biologically active
variant of that sequence.
Obtaining Full-Length Polynucleotides
[0059] The partial sequence of SEQ ID NO: 1, 2, 3, 4, 5, or 6 or its complement can be used
to identify the corresponding full length gene from which they were derived. The partial
sequences can be nick-translated or end-labeled with
32P using polynucleotide kinase using labeling methods known to those with skill in
the art (
BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al., eds., Elsevier Press, N.Y., 1986). A lambda library prepared from human tissue can be directly screened with the labeled
sequences of interest or the library can be converted en masse to pBluescript (Stratagene
Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening
(see Sambrook
et al., 1989, pg. 1.20).
[0060] Both methods are well known in the art. Briefly, filters with bacterial colonies
containing the library in pBluescript or bacterial lawns containing lambda plaques
are denatured, and the DNA is fixed to the filters. The filters are hybridized with
the labeled probe using hybridization conditions described by Davis
et al., 1986. The partial sequences, cloned into lambda or pBluescript, can be used as
positive controls to assess background binding and to adjust the hybridization and
washing stringencies necessary for accurate clone identification. The resulting radiographies
are compared to duplicate plates of colonies or plaques; each exposed spot corresponds
to a positive colony or plaque. The colonies or plaques are selected and expanded,
and the DNA is isolated from the colonies for further analysis and sequencing.
[0061] Positive cDNA clones are analyzed to determine the amount of additional sequence
they contain using PCR with one primer from the partial sequence and the other primer
from the vector. Clones with a larger vector-insert PCR product than the original
partial sequence are analyzed by restriction digestion and DNA sequencing to determine
whether they contain an insert of the same size or similar as the mRNA size determined
from Northern blot Analysis.
[0062] Once one or more overlapping cDNA clones are identified, the complete sequence of
the clones can be determined, for example after exonuclease III digestion (
McCombie et al., Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting
overlapping sequences are assembled into a single contiguous sequence of high redundancy
(usually three to five overlapping sequences at each nucleotide position), resulting
in a highly accurate final sequence.
[0063] Various PCR-based methods can be used to extend the nucleic acid sequences encoding
the disclosed portions of human RC Kinase to detect upstream sequences such as promoters
and regulatory elements. For example, restriction-site PCR uses universal primers
to retrieve unknown sequence adjacent to a known locus (
Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence
and a primer specific to the known region. The amplified sequences are then subjected
to a second round of PCR with the same linker primer and another specific primer internal
to the first one. Products of each round of PCR are transcribed with an appropriate
RNA polymerase and sequenced using reverse transcriptase.
[0064] Inverse PCR also can be used to amplify or extend sequences using divergent primers
based on a known region (
Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06
Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30
nucleotides in length, to have a GC content of 50% or more, and to anneal to the target
sequence at temperatures about 68 - 72°C. The method uses several restriction enzymes
to generate a suitable fragment in the known region of a gene. The fragment is then
circularized by intramolecular ligation and used as a PCR template.
[0065] Another method which can be used is capture PCR, which involves PCR amplification
of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome
DNA (
Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations are used to
place an engineered double-stranded sequence into an unknown fragment of the DNA molecule
before performing PCR.
[0066] Another method which can be used to retrieve unknown sequences is that of
Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991. Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo
Alto, Calif.) can be used to walk genomic DNA. This process avoids the need to screen
libraries and is useful in finding intron/exon junctions.
[0067] When screening for full-length cDNAs, it is preferable to use libraries that have
been size-selected to include larger cDNAs. Also, random-primed libraries are preferable,
in that they will contain more sequences which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for situations in which
an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be
useful for extension of sequence into 5' non-transcribed regulatory regions.
[0068] Commercially available capillary electrophoresis systems can be used to analyze the
size or confirm the nucleotide sequence of PCR or sequencing products. For example,
capillary sequencing can employ flowable polymers for electrophoretic separation,
four different fluorescent dyes (one for each nucleotide) which are laser activated,
and detection of the emitted wavelengths by a charge coupled device camera. Output/light
intensity can be converted to electrical signal using appropriate software (
e.
g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading
of samples to computer analysis and electronic data display can be computer controlled.
Capillary electrophoresis is especially preferable for the sequencing of small pieces
of DNA which might be present in limited amounts in a particular sample.
Obtaining Polypeptides
[0069] RC Kinase polypeptides can be obtained, for example, by purification from human cells,
by expression of RC Kinase polynucleotides, or by direct chemical synthesis.
Protein Purification
[0070] RC Kinase polypeptides can be purified from cells, including cells which have been
transfected with RC Kinase expression constructs. Kidney, fetal lung, testis, B cells,
adult lung epithelium, and chronic lymphatic leukemia cells are particularly useful
sources of RC Kinase polypeptides. A purified RC Kinase polypeptide is separated from
other compounds which normally associate with the RC Kinase polypeptide in the cell,
such as certain proteins, carbohydrates, or lipids, using methods well-known in the
art. Such methods include, but are not limited to, size exclusion chromatography,
ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography,
and preparative gel electrophoresis. A preparation of purified RC Kinase polypeptides
is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity
of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide
gel electrophoresis. Enzymatic activity of the purified preparations can be assayed,
for example, as described in Example 2.
Expression of Polynucleotides
[0071] To express a RC Kinase polypeptide, a RC Kinase polynucleotide can be inserted into
an expression vector which contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods which are well known to those
skilled in the art can be used to construct expression vectors containing sequences
encoding RC Kinase polypeptides and appropriate transcriptional and translational
control elements. These methods include
in vitro recombinant DNA techniques, synthetic techniques, and
in vivo genetic recombination. Such techniques are described, for example, in Sambrook
et al. (1989) and
Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York,
N.Y, 1989.
[0072] A variety of expression vector/host systems can be utilized to contain and express
sequences encoding a RC Kinase polypeptide. These include, but are not limited to,
microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid,
or cosmid DNA expression vectors; yeast transformed with yeast expression vectors,
insect cell systems infected with virus expression vectors (
e.
g., baculovirus), plant cell systems transformed with virus expression vectors (
e.
g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression
vectors (
e.
g., Ti or pBR322 plasmids), or animal cell systems.
[0073] The control elements or regulatory sequences are those non-translated regions of
the vector - enhancers, promoters, 5' and 3' untranslated regions -- which interact
with host cellular proteins to carry out transcription and translation. Such elements
can vary in their strength and specificity. Depending on the vector system and host
utilized, any number of suitable transcription and translation elements, including
constitutive and inducible promoters, can be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid
(Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like
can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters
or enhancers derived from the genomes of plant cells (
e.
g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (
e.
g., viral promoters or leader sequences) can be cloned into the vector. In mammalian
cell systems, promoters from mammalian genes or from mammalian viruses are preferable.
If it is necessary to generate a cell line that contains multiple copies of a nucleotide
sequence encoding a RC Kinase polypeptide, vectors based on SV40 or EBV can be used
with an appropriate selectable marker.
Bacterial and Yeast Expression Systems
[0074] In bacterial systems, a number of expression vectors can be selected depending upon
the use intended for the RC Kinase polypeptide. For example, when a large quantity
of a RC Kinase polypeptide is needed for the induction of antibodies, vectors which
direct high level expression of fusion proteins that are readily purified can be used.
Such vectors include, but are not limited to, multifunctional
E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the RC Kinase polypeptide can be ligated into the vector in frame with sequences
for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that
a hybrid protein is produced. pIN vectors (
Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989 or pGEX vectors (Promega, Madison, Wis.) can be used to express foreign polypeptides
as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins
are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione. Proteins made in such
systems can be designed to include heparin, thrombin, or Factor Xa protease cleavage
sites so that the cloned polypeptide of interest can be released from the GST moiety
at will.
[0075] In the yeast
Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel
et al. (1989) and
Grant et al., Methods Enzymol. 153, 516-544, 1987.
Plant and Insect Expression Systems
[0076] If plant expression vectors are used, the expression of sequences encoding RC Kinase
polypeptides can be driven by any of a number of promoters. For example, viral promoters
such as the 35S and 19S promoters of CaMV can be used alone or in combination with
the omega leader sequence from TMV (
Takamatsu EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock
promoters can be used (
Coruzzi et al., EMBO J. 3, 1671-1680, 1984;
Broglie et al., Science 224, 838-843, 1984;
Winter et al., Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation
or by pathogen-mediated transfection. Such techniques are described in a number of
generally available reviews (see, for example,
Hobbs or Murray, in McGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New
York, N.Y., pp. 191-196, 1992).
[0077] An insect system also can be used to express a RC Kinase polypeptide. For example,
in one such system
Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in
Spodoptera frugiperda cells or in
Trichoplusia larvae. Sequences encoding RC Kinase polypeptides can be cloned into a non-essential
region of the virus, such as the polyhedrin gene, and placed under control of the
polyhedrin promoter. Successful insertion of RC Kinase polypeptides will render the
polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant
viruses can then be used to infect, for example,
S. frugiperda cells or
Trichoplusia larvae in which RC Kinase polypeptides can be expressed (
Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).
Mammalian Expression Systems
[0078] A number of viral-based expression systems can be utilized in mammalian host cells.
For example, if an adenovirus is used as an expression vector, sequences encoding
RC Kinase polypeptides can be ligated into an adenovirus transcription/translation
complex consisting of the late promoter and tripartite leader sequence. Insertion
in a non-essential E1 or E3 region of the viral genome can be used to obtain a viable
virus which is capable of expressing a RC Kinase polypeptide in infected host cells
(
Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,
can be used to increase expression in mammalian host cells.
[0079] Human artificial chromosomes (HACs) also can be used to deliver larger fragments
of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed
and delivered to cells via conventional delivery methods (
e.g., liposomes, polycationic amino polymers, or vesicles).
[0080] Specific initiation signals also can be used to achieve more efficient translation
of sequences encoding RC Kinase polypeptides. Such signals include the ATG initiation
codon and adjacent sequences. In cases where sequences encoding a RC Kinase polypeptide,
its initiation codon, and upstream sequences are inserted into the appropriate expression
vector, no additional transcriptional or translational control signals may be needed.
However, in cases where only coding sequence, or a fragment thereof, is inserted,
exogenous translational control signals (including the ATG initiation codon) should
be provided. The initiation codon should be in the correct reading frame to ensure
translation of the entire insert. Exogenous translational elements and initiation
codons can be of various origins, both natural and synthetic. The efficiency of expression
can be enhanced by the inclusion of enhancers which are appropriate for the particular
cell system which is used (see
Scharf et al., Results Probl. Cell Differ. 20, 125-162, 1994).
[0081] The invention relates to an expression vector containing any polynucleotide according
to the invention.
Host Cells
[0082] A host cell strain can be chosen for its ability to modulate the expression of the
inserted sequences or to process an expressed RC Kinase polypeptide in the desired
fashion. Such modifications of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational
processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate
correct insertion, folding and/or function. Different host cells which have specific
cellular machinery and characteristic mechanisms for post-translational activities
(
e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture
Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be
chosen to ensure the correct modification and processing of the foreign protein.
[0083] Stable expression is preferred for long-term, high-yield production of recombinant
proteins. For example, cell lines which stably express RC Kinase polypeptides can
be transformed using expression vectors which can contain viral origins of replication
and/or endogenous expression elements and a selectable marker gene on the same or
on a separate vector. Following the introduction of the vector, cells can be allowed
to grow for 1-2 days in an enriched medium before they are switched to a selective
medium. The purpose of the selectable marker is to confer resistance to selection,
and its presence allows growth and recovery of cells which successfully express the
introduced RC Kinase sequences. Resistant clones of stably transformed cells can be
proliferated using tissue culture techniques appropriate to the cell type.
[0084] Any number of selection systems can be used to recover transformed cell lines. These
include, but are not limited to, the herpes simplex virus thymidine kinase (
Wigler et al., Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (
Lowy et al., Cell 22, 817-23, 1980). Genes which can be employed in
tk- or
aprt- cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can
be used as the basis for selection. For example,
dhfr confers resistance to methotrexate (
Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980);
npt confers resistance to the aminoglycosides, neomycin and G-418 (
Colbere-Garapin et al., J. Mol. Biol. 150, 1-14, 1981); and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase,
respectively 5 (Murray, 1992 supra). Additional selectable genes have been described,
for example
trpB, which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine (
Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, 13-glucuronidase and its substrate GUS, and
luciferase and its substrate luciferin, can be used to identify transformants and
to quantify the amount of transient or 10 stable protein expression attributable to
a specific vector system (
Rhodes et al., Methods Mol. Biol. 55, 121-131, 1995).
Detecting Expression of Polypeptides
[0085] The invention relates to a method for detection of a polynucleotide encoding a RC
Kinase polypeptide according to the invention in a biological sample comprising the
following steps:
- (a) hybridizing any polynucleotide of the invention to a nucleic acid material of
a biological sample, thereby forming a hybridization complex; and
- (b) detecting said hybridization complex.
[0086] The invention also relates to the method as described above, wherein before hybridization,
the nucleic acid material of the biological sample is amplified.
[0087] The invention also relates to a method for the detection of a polynucleotide of the
invention or a RC Kinase polypeptide of of the invention comprising contacting a biological
sample with a reagent which specifically interacts with the polynucleotide or the
polypeptide, wherein the reagent is a ribozyme, an antisense oligonucleotide or an
antibody.
[0088] The invention also relates to a diagnostic kit for conducting any of the method as
described above.
[0089] Although the presence of marker gene expression suggests that the RC Kinase polynucleotide
is also present, its presence and expression may need to be conformed. For example,
if a sequence encoding a RC Kinase polypeptide is inserted within a marker gene sequence,
transformed cells containing sequences which encode a RC Kinase polypeptide can be
identified by the absence of marker gene function. Alternatively, a marker gene can
be placed in tandem with a sequence encoding a RC Kinase polypeptide under the control
of a single promoter. Expression of the marker gene in response to induction or selection
usually indicates expression of the RC Kinase polynucleotide.
[0090] Alternatively, host cells which contain a RC Kinase polynucleotide and which express
a RC Kinase polypeptide can be identified by a variety of procedures known to those
of skill in the art. These procedures include, but are not limited to, DNA-DNA or
DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include
membrane, solution, or chip-based technologies for the detection and/or quantification
of nucleic acid or protein.
[0091] The presence of a polynucleotide sequence encoding a RC Kinase polypeptide can be
detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments
or fragments of polynucleotides encoding a RC Kinase polypeptide. Nucleic acid amplification-based
assays involve the use of oligonucleotides selected from sequences encoding a RC Kinase
polypeptide to detect transformants which contain a RC Kinase polynucleotide.
[0092] A variety of protocols for detecting and measuring the expression of a RC Kinase
polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide,
are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering
epitopes on a RC Kinase polypeptide can be used, or a competitive binding assay can
be employed. These and other assays are described in
Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn.,
1990) and
Maddox et al., J. Exp. Med. 158, 1211-1216, 1983).
[0093] A wide variety of labels and conjugation techniques are known by those skilled in
the art and can be used in various nucleic acid and amino acid assays. Means for producing
labeled hybridization or PCR probes for detecting sequences related to polynucleotides
encoding RC Kinase polypeptides include oligolabeling, nick translation, end-labeling,
or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding
a RC Kinase polypeptide can be cloned into a vector for the production of an mRNA
probe. Such vectors are known in the art, are commercially available, and can be used
to synthesize RNA probes
in vitro by addition of labeled nucleotides and an appropriate RNA polymerase, such as T7,
T3, or SP6. These procedures can be conducted using a variety of commercially available
kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter
molecules or labels which can be used for ease of detection include radionuclides,
enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0094] The invention relates to a method for producing a RC Kinase polypeptide of the invention,
wherein the method comprises the following steps: (a) culturing the host cell of the
invention under conditions suitable for the expression of the polypeptide; and (b)
recovering the polypeptide from the host cell culture.
Expression and Purification of Polypeptides
[0095] Host cells transformed with nucleotide sequences encoding a RC Kinase polypeptide
can be cultured under conditions suitable for the expression and recovery of the protein
from cell culture. The polypeptide produced by a transformed cell can be secreted
or contained intracellularly depending on the sequence and/or the vector used. As
will be understood by those of skill in the art, expression vectors containing polynucleotides
which encode RC Kinase polypeptides can be designed to contain signal sequences which
direct secretion of RC Kinase polypeptides through a prokaryotic or eukaryotic cell
membrane.
[0096] Other constructions can be used to join a sequence encoding a RC Kinase polypeptide
to a nucleotide sequence encoding a polypeptide domain which will facilitate purification
of soluble proteins. Such purification facilitating domains include, but are not limited
to, metal chelating peptides such as histidine-tryptophan modules that allow purification
on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin,
and the domain utilized in the FLAGS extension/affinity purification system (Immunex
Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those
specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification
domain and the RC Kinase polypeptide can be used to facilitate purification. One such
expression vector provides for expression of a fusion protein containing a RC Kinase
polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage
site. The histidine residues facilitate purification on IMAC (immobilized metal ion
affinity chromatography as described in
Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the RC Kinase
polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed
in
Kroll et al., DNA Cell Biol. 12, 441-453, 1993).
Chemical Synthesis
[0097] Sequences encoding a RC Kinase polypeptide can be synthesized, in whole or in part,
using chemical methods well known in the art (see
Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223, 1980;
Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a RC Kinase polypeptide itself can be produced using chemical methods
to synthesize its amino acid sequence. For example, RC Kinase polypeptides can be
produced by direct peptide synthesis using solid-phase techniques (
Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963;
Roberge et al., Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated
synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer
(Perkin Elmer). Various fragments of RC Kinase polypeptides can be separately synthesized
and combined using chemical methods to produce a full-length molecule.
[0098] The newly synthesized peptide can be substantially purified by preparative high performance
liquid chromatography (
e.
g.,
Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New
York, N.Y., 1983). The composition of a synthetic RC Kinase polypeptide can be confirmed by amino
acid analysis or sequencing (
e.
g., the Edman degradation procedure;
see Creighton,
supra). Additionally, any portion of the amino acid sequence of the RC Kinase polypeptide
can be altered during direct synthesis and/or combined using chemical methods with
sequences from other proteins to produce a variant polypeptide or a fusion protein.
Production of Altered Polypeptides
[0099] As will be understood by those of skill in the art, it may be advantageous to produce
RC Kinase polypeptide-encoding nucleotide sequences possessing non-naturally occurring
codons. For example, codons preferred by a particular prokaryotic or eukaryotic host
can be selected to increase the rate of protein expression or to produce an RNA transcript
having desirable properties, such as a half-life which is longer than that of a transcript
generated from the naturally occurring sequence.
[0100] The nucleotide sequences disclosed herein can be engineered using methods generally
known in the art to alter RC Kinase polypeptide-encoding sequences for a variety of
reasons, including modification of the cloning, processing, and/or expression of the
gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments
and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For
example, site-directed mutagenesis can be used to insert new restriction sites, alter
glycosylation patterns, change codon preference, produce splice variants, introduce
mutations, and so forth.
Antibodies
[0101] Any type of antibody known in the art can be generated to bind specifically to an
epitope of a RC Kinase polypeptide. "Antibody" as used herein includes intact immunoglobulin
molecules, as well as fragments thereof, such as Fab, F(ab')
2, and Fv, which are capable of binding an epitope of a RC Kinase polypeptide. Typically,
at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However,
epitopes which involve non-contiguous amino acids may require more,
e.
g., at least 15, 25, or 50 amino acids.
[0102] An antibody which specifically binds to an epitope of a RC Kinase polypeptide can
be used therapeutically, as well as in immunochemical assays, including but not limited
to Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations,
or other immunochemical assays known in the art. Various immunoassays can be used
to identify antibodies having the desired specificity. Numerous protocols for competitive
binding or immuno-radiometric assays are well known in the art. Such immunoassays
typically involve the measurement of complex formation between an immunogen and an
antibody which specifically binds to the immunogen.
[0103] Typically, an antibody which specifically binds to a RC Kinase polypeptide provides
a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided
with other proteins when used in an immunochemical assay. Preferably, antibodies which
specifically bind to RC Kinase polypeptides do not detect other proteins in immunochemical
assays and can immunoprecipitate a RC Kinase polypeptide from solution.
[0104] RC Kinase polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit,
guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a RC Kinase
polypeptide can be conjugated to a carrier protein, such as bovine serum albumin,
thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various
adjuvants can be used to increase the immunological response. Such adjuvants include,
but are not limited to, Freund's adjuvant, mineral gels (
e.
g., aluminum hydroxide), and surface active substances (
e.
g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (
bacilli Calmette-Guerin) and
Corynebacterium parvum are especially useful.
[0105] Monoclonal antibodies which specifically bind to a RC Kinase polypeptide can be prepared
using any technique which provides for the production of antibody molecules by continuous
cell lines in culture. These techniques include, but are not limited to, the hybridoma
technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (
Kohler et al., Nature 256, 495-497, 1985;
Kozbor et al., J. Immunol. Methods 81, 31-42, 1985;
Cote et al., Proc. Natl. Acad. Sci. 80, 2026-2030, 1983;
Cole et al., Mol. Cell Biol. 62, 109-120, 1984).
[0106] In addition, techniques developed for the production of "chimeric antibodies," the
splicing of mouse antibody genes to human antibody genes to obtain a molecule with
appropriate antigen specificity and biological activity, can be used (
Morrison et al., Proc. Natl. Acad. Sci. 81, 6851-6855, 1984;
Neuberger et al., Nature 312, 604-608, 1984;
Takeda et al., Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from
mounting an immune response against the antibody when it is used therapeutically.
Such antibodies may be sufficiently similar in sequence to human antibodies to be
used directly in therapy or may require alteration of a few key residues. Sequence
differences between rodent antibodies and human sequences can be minimized by replacing
residues which differ from those in the human sequences by site directed mutagenesis
of individual residues or by grating of entire complementarity determining regions.
Alternatively, one can produce humanized antibodies using recombinant methods, as
described in
GB2188638B. Antibodies which specifically bind to a RC Kinase polypeptide can contain antigen
binding sites which are either partially or fully humanized, as disclosed in
U.S. 5,565,332.
[0107] Alternatively, techniques described for the production of single chain antibodies
can be adapted using methods known in the art to produce single chain antibodies which
specifically bind to RC Kinase polypeptides. Antibodies with related specificity,
but of distinct idiotypic composition, can be generated by chain shuffling from random
combinatorial immunoglobin libraries (
Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).
[0108] Single-chain antibodies also can be constructed using a DNA amplification method,
such as PCR, using hybridoma cDNA as a template (
Thirion et al., 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent.
Construction of tetravalent, bispecific single-chain antibodies is taught, for example,
in
Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in
Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.
[0109] A nucleotide sequence encoding a single-chain antibody can be constructed using manual
or automated nucleotide synthesis, cloned into an expression construct using standard
recombinant DNA methods, and introduced into a cell to express the coding sequence,
as described below. Alternatively, single-chain antibodies can be produced directly
using, for example, filamentous phage technology.
Verhaar et al., 1995, Int. J. Cancer 61, 497-501;
Nicholls et al., 1993, J. Immunol. Meth. 165, 81-91.
[0111] Other types of antibodies can be constructed and used therapeutically in methods
of the invention. For example, chimeric antibodies can be constructed as disclosed
in
WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent
and multispecific, such as the "diabodies" described in
WO 94/13804, also can be prepared.
[0112] Antibodies disclosed herein can be purified by methods well known in the art. For
example, antibodies can be affinity purified by passage over a column to which a RC
Kinase polypeptide is bound. The bound antibodies can then be eluted from the column
using a buffer with a high salt concentration.
Antisense Oligonucleotides
[0113] Antisense oligonucleotides are nucleotide sequences which are complementary to a
specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides
combine with natural sequences produced by the cell to form complexes and block either
transcription or translation. Preferably, an antisense oligonucleotide is at least
11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50
or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide
molecules can be provided in a DNA construct and introduced into a cell as described
above to decrease the level of RC Kinase gene products in the cell.
[0114] Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination
of both. Oligonucleotides can be synthesized manually or by an automated synthesizer,
by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide
with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates,
phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates,
phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate
triesters.
See Brown, Meth. Mol. Biol. 20, 1-8, 1994;
Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994;
Uhlmann et al., Chem. Rev. 90, 543-583, 1990.
[0115] Modifications of RC Kinase gene expression can be obtained by designing antisense
oligonucleotides which will form duplexes to the control, 5', or regulatory regions
of the RC Kinase gene. Oligonucleotides derived from the transcription initiation
site,
e.
g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition
can be achieved using "triple helix" base-pairing methodology. Triple helix pairing
is useful because it causes inhibition of the ability of the double helix to open
sufficiently for the binding of polymerases, transcription factors, or chaperons.
Therapeutic advances using triplex DNA have been described in the literature (e.g.,
Gee et al., in Huber & Carr, MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing
Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA
by preventing the transcript from binding to ribosomes.
[0116] Precise complementarity is not required for successful duplex formation between an
antisense oligonucleotide and the complementary sequence of a RC Kinase polynucleotide.
Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches
of contiguous nucleotides which are precisely complementary to a RC Kinase polynucleotide,
each separated by a stretch of contiguous nucleotides which are not complementary
to adjacent RC Kinase nucleotides, can provide targeting specificity for RC Kinase
mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least
4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences
are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily
use the calculated melting point of an antisense-sense pair to determine the degree
of mismatching which will be tolerated between a particular antisense oligonucleotide
and a particular RC Kinase polynucleotide sequence.
[0117] Antisense oligonucleotides can be modified without affecting their ability to hybridize
to a RC Kinase polynucleotide. These modifications can be internal or at one or both
ends of the antisense molecule. For example, internucleoside phosphate linkages can
be modified by adding cholesteryl or diamine moieties with varying numbers of carbon
residues between the amino groups and terminal ribose. Modified bases and/or sugars,
such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which
the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed
in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared
by methods well known in the art.
See, e.
g.,
Agrawal et al., Trends Biotechnol. 10, 152-158, 1992;
Uhlmann et al., Chem. Rev. 90, 543-584, 1990;
Uhlmann et al., Tetrahedron. Lett. 215, 3539-3542, 1987.
Ribozymes
[0118] Ribozymes are RNA molecules with catalytic activity.
See, e.g., Cech, Science 236, 1532-1539; 1987;
Cech, Ann. Rev. Biochem. 59, 543-568; 1990,
Cech, Curr. Opin. Struct. Biol. 2, 605-609; 1992,
Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is
known in the art (
e.
g.,
Haseloff et al., U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the
ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
Examples include engineered hammerhead motif ribozyme molecules that can specifically
and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
[0119] The coding sequence of a RC Kinase polynucleotide can be used to generate ribozymes
which will specifically bind to mRNA transcribed from the RC Kinase polynucleotide.
Methods of designing and constructing ribozymes which can cleave other RNA molecules
in trans in a highly sequence specific manner have been developed and described in
the art (
see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs
by engineering a discrete "hybridization" region into the ribozyme. The hybridization
region contains a sequence complementary to the target RNA and thus specifically hybridizes
with the target (see, for example,
Gerlach et al., EP 321,201).
[0120] Specific ribozyme cleavage sites within a RC Kinase RNA target are initially identified
by scanning the RNA molecule for ribozyme cleavage sites which include the following
sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and
20 ribonucleotides corresponding to the region of the RC Kinase target RNA containing
the cleavage site can be evaluated for secondary structural features which may render
the target inoperable. The suitability of candidate targets also can be evaluated
by testing accessibility to hybridization with complementary oligonucleotides using
ribonuclease protection assays. Longer complementary sequences can be used to increase
the affinity of the hybridization sequence for the target. The hybridizing and cleavage
regions of the ribozyme can be integrally related; thus, upon hybridizing to the RC
Kinase target RNA through the complementary regions, the catalytic region of the ribozyme
can cleave the target.
[0121] Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods,
such as microinjection, liposome-mediated transfection, electroporation, or calcium
phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct
into cells in which it is desired to decrease RC Kinase expression. Alternatively,
if it is desired that the cells stably retain the DNA construct, it can be supplied
on a plasmid and maintained as a separate element or integrated into the genome of
the cells, as is known in the art. The DNA construct can include transcriptional regulatory
elements, such as a promoter element, an enhancer or UAS element, and a transcriptional
terminator signal, for controlling transcription of ribozymes in the cells.
[0122] As taught in
Haseloff et al., U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to
factors which induce expression of a target gene. Ribozymes also can be engineered
to provide an additional level of regulation, so that destruction of RC Kinase mRNA
occurs only when both a ribozyme and a target gene are induced in the cells.
Screening Methods
[0123] The invention provides methods for identifying modulators,
i.
e., candidate or test compounds which bind to RC Kinase polypeptides or polynucleotides
and/or have a stimulatory or inhibitory effect on, for example, expression or activity
of the RC Kinase polypeptide or polynucleotide, so as to regulate signaling through
a MAP kinase phosphorelay system, regulate the production of inflammatory mediators,
and regulate degradation of the extracellular matrix. Decreased signaling through
a MAP kinase phosphorelay system and decreased production of inflammatory mediators
are useful for preventing undesired inflammation, cell proliferation, cell differentiation,
cytokine production, or apoptosis. Increased signaling through a MAP kinase phosphorelay
system and increased production of inflammatory mediators is useful for enhancing
the repair of damaged tissues, enhancing resistance to irritants or toxins, or increasing
the resistance to infection. Decreased extracellular matrix degradation is useful
for preventing damage or irreversible changes to lung microstructures and preventing
or suppressing malignant cells from metastasizing. Increased extracellular matrix
degradation may be desired, for example, in developmental disorders characterized
by inappropriately low levels of extracellular matrix degradation or in regeneration.
[0124] The invention provides assays for screening test compounds which bind to or modulate
the activity of a RC Kinase polypeptide or a RC Kinase polynucleotide. A test compound
preferably binds to a RC Kinase polypeptide or polynucleotide. More preferably, a
test compound decreases a RC Kinase activity of a RC Kinase polypeptide or expression
of a RC Kinase polynucleotide by at least about 10, preferably about 50, more preferably
about 75, 90, or 100% relative to the absence of the test compound.
[0125] The invention comprises a method of screening for agents which decrease the activity
of a RC Kinase polypeptide according to the invention, comprising the steps of: (a)
contacting a test compound with a polypeptide encoded by any polynucleotide of the
invention; and (b) detecting binding of the test compound to the polypeptide, wherein
a test compound which binds to the polypeptide is identified as a potential therapeutic
agent for decreasing the activity of said polypeptide.
[0126] Further, a method of screening for agents which regulate the activity of a RC Kinase
polypeptide; comprising the steps of : (a) contacting a test compound with a RC Kinase
polypeptide encoded by any polynucleotide of the invention; and (b) detecting phosphorylation
activity of the RC Kinase polypeptide, wherein a test compound which increases the
activity of the polypeptide is identified as a potential therapeutic agent for increasing
the activity of the RC Kinase polypeptide, and wherein a test compound which decreases
the activity of the polypeptide is identified as a potential therapeutic agent for
decreasing the activity of the RC Kinase polypeptide is encompassed in the invention.
[0127] The invention also relates to a method of screening for agents which regulate the
activity of a RC Kinase, comprising the steps of: (a) contacting a test compound with
a RC Kinase polypeptide encoded by any polynucleotide of the invention as described
above and MKK4; and (b) detecting RC Kinase polypeptide phosphorylation of MKK4, wherein
a test compound which increases the RC Kinase polypeptide phosphorylation of MKK4
is identified as a potential therapeutic agent for increasing the activity of the
RC Kinase polypeptide, and wherein a test compound which decreases the RC Kinase polypeptide
phosphorylation of MKK4 is identified as a potential therapeutic agent for decreasing
the activity of the RC Kinase polypeptide. Also, a method of screening for agents
which decrease the activity of a RC Kinase polypeptide, comprising contacting a test
compound with any polynucleotide of the invention and detecting binding of the test
compound to the polynucleotide, wherein a test compound which binds to the polynucleotide
is identified as a potential therapeutic agent for decreasing the activity of RC Kinase
polypeptide is encompassed by the invention.
Test Compounds
[0128] Test compounds can be pharmacologic agents already known in the art or can be compounds
previously unknown to have any pharmacological activity. The compounds can be naturally
occurring or designed in the laboratory. They can be isolated from microorganisms,
animals, or plants, and can be produced recombinantly, or synthesized by chemical
methods known in the art. If desired, test compounds can be obtained using any of
the numerous combinatorial library methods known in the art, including but not limited
to, biological libraries, spatially addressable parallel solid phase or solution phase
libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound"
library method, and synthetic library methods using affinity chromatography selection.
The biological library approach is limited to polypeptide libraries, while the other
four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule
libraries of compounds.
See Lam, Anticancer Drug Des. 12, 145, 1997.
[0129] Methods for the synthesis of molecular libraries are well known in the art (
see, for example,
DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993;
Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994;
Zuckermann et al., J. Med. Chem. 37, 2678, 1994;
Cho et al., Science 261, 1303, 1993;
Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2059, 1994;
Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061;
Gallop et al., J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (
see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (
Lam, Nature 354, 82-84, 1991), chips (
Fodor, Nature 364, 555-556, 1993), bacteria or spores (
Ladner, U.S. Patent 5,223,409), plasmids (
Cull et al., Proc. Natl. Acad. Sci. U.S.A. 89, 1865-1869, 1992), or phage (
Scott & Smith, Science 249, 386-390, 1990;
Devlin, Science 249, 404-406, 1990);
Cwirla et al., Proc. Natl. Acad. Sci. 97, 6378-6382, 1990;
Felici, J. Mol. Biol. 222, 301-310, 1991; and
Ladner, U.S. Patent 5,223,409).
High Throughput Screening
[0130] Test compounds can be screened for the ability to bind to RC Kinase polypeptides
or polynucleotides or to affect RC Kinase activity or RC Kinase gene expression using
high throughput screening. Using high throughput screening, many discrete compounds
can be tested in parallel so that large numbers of test compounds can be quickly screened.
The most widely established techniques utilize 96-well microtiter plates. The wells
of the microtiter plates typically require assay volumes that range from 50 to 500
µl. In addition to the plates, many instruments, materials, pipettors, robotics, plate
washers, and plate readers are commercially available to fit the 96-well format.
[0131] Alternatively, "free format assays," or assays that have no physical barrier between
samples, can be used. For example, an assay using pigment cells (melanocytes) in a
simple homogeneous assay for combinatorial peptide libraries is described by
Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial
compounds are placed on the surface of the agarose. The combinatorial compounds are
partially released the compounds from the beads. Active compounds can be visualized
as dark pigment areas because, as the compounds diffuse locally into the gel matrix,
the active compounds cause the cells to change colors.
[0134] Another high throughput screening method is described in
Beutel et al., U.S. Patent 5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components
are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic
sheet, a filter, or other form of easily manipulated solid support. When samples are
introduced to the porous matrix they diffuse sufficiently slowly, such that the assays
can be performed without the test samples running together.
Binding Assays
[0135] For binding assays, the test compound is preferably a small molecule which binds
to and occupies the active site or a fibronectin domain of the RC Kinase polypeptide,
thereby making the active site or fibronectin domain inaccessible to substrate such
that normal biological activity is prevented. Examples of such small molecules include,
but are not limited to, small peptides or peptide-like molecules. In binding assays,
either the test compound or the RC Kinase polypeptide can comprise a detectable label,
such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as
horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound
which is bound to the RC Kinase polypeptide can then be accomplished, for example,
by direct counting of radioemmission, by scintillation counting, or by determining
conversion of an appropriate substrate to a detectable product.
[0136] Alternatively, binding of a test compound to a RC Kinase polypeptide can be determined
without labeling either of the interactants. For example, a microphysiometer can be
used to detect binding of a test compound with a target polypeptide. A microphysiometer
(
e.
g., Cytosensor™) is an analytical instrument that measures the rate at which a cell
acidifies its environment using a light-addressable potentiometric sensor (LAPS).
Changes in this acidification rate can be used as an indicator of the interaction
between a test compound and a RC Kinase polypeptide. (
McConnell et al., Science 257, 1906-1912, 1992).
[0137] Determining the ability of a test compound to bind to a RC Kinase polypeptide also
can be accomplished using a technology such as real-time Bimolecular Interaction Analysis
(BIA).
Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and
Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705, 1995. BIA is a technology for studying biospecific interactions in real time, without
labeling any of the interactants (
e.
g., BIAcore
™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as
an indication of real-time reactions between biological molecules.
[0138] In yet another aspect of the invention, a RC Kinase polypeptide can be used as a
"bait protein" in a two-hybrid assay or three-hybrid assay (see,
e.g., U.S. Patent 5,283,317;
Zervos et al., Cell 72, 223-232, 1993;
Madura et al., J. Biol. Chem. 268, 12046-12054, 1993;
Bartel et al., BioTechniques 14, 920-924, 1993;
Iwabuchi et al., Oncogene 8, 1693-1696, 1993; and
Brent WO94/10300), to identify other proteins which bind to or interact with the RC Kinase polypeptide
and modulate its activity.
[0139] The two-hybrid system is based on the modular nature of most transcription factors,
which consist of separable DNA-binding and activation domains. Briefly, the assay
utilizes two different DNA constructs. For example, in one construct a polynucleotide
encoding a RC Kinase polypeptide is fused to a polynucleotide encoding the DNA binding
domain of a known transcription factor (
e.
g., GAL-4). In the other construct, a DNA sequence that encodes an unidentified protein
("prey" or "sample") is fused to a polynucleotide that codes for the activation domain
of the known transcription factor. If the "bait" and the "prey" proteins are able
to interact
in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the
transcription factor are brought into close proximity. This proximity allows transcription
of a reporter gene (
e.
g., LacZ), which is operably linked to a transcriptional regulatory site responsive
to the transcription factor. Expression of the reporter gene can be detected, and
cell colonies containing the functional transcription factor can be isolated and used
to obtain the DNA sequence encoding the protein which interacts with the RC Kinase
polypeptide.
[0140] It may be desirable to immobilize either the RC Kinase polypeptide (or polynucleotide)
or the test compound to facilitate separation of bound from unbound forms of one or
both of the interactants, as well as to accommodate automation of the assay. Thus,
either the RC Kinase polypeptide (or polynucleotide) or the test compound can be bound
to a solid support. Suitable solid supports include, but are not limited to, glass
or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips,
or particles such as beads (including, but not limited to, latex, polystyrene, or
glass beads). Any method known in the art can be used to attach the RC Kinase polypeptide
(or polynucleotide) or test compound to a solid support, including use of covalent
and non-covalent linkages, passive absorption, or pairs of binding moieties attached
respectively to the polypeptide or test compound and the solid support. Test compounds
are preferably bound to the solid support in an array, so that the location of individual
test compounds can be tracked. Binding of a test compound to a RC Kinase polypeptide
(or polynucleotide) can be accomplished in any vessel suitable for containing the
reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge
tubes.
[0141] In one embodiment, a RC Kinase polypeptide is a fusion protein comprising a domain
that allows the RC Kinase polypeptide to be bound to a solid support. For example,
glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose
beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates,
which are then combined with the test compound or the test compound and the non-adsorbed
RC Kinase polypeptide; the mixture is then incubated under conditions conducive to
complex formation (
e.
g., at physiological conditions for salt and pH). Following incubation, the beads or
microtiter plate wells are washed to remove any unbound components. Binding of the
interactants can be determined either directly or indirectly, as described above.
Alternatively, the complexes can be dissociated from the solid support before binding
is determined.
[0142] Other techniques for immobilizing polypeptides or polynucleotides on a solid support
also can be used in the screening assays of the invention. For example, either a RC
Kinase polypeptide (or polynucleotide) or a test compound can be immobilized utilizing
conjugation of biotin and streptavidin. Biotinylated RC Kinase polypeptides or test
compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well
known in the art (
e.
g., biotinylation kit, Pierce Chemicals, Rockford, III.) and immobilized in the wells
of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies
which specifically bind to a RC Kinase polypeptide polynucleotides, or a test compound,
but which do not interfere with a desired binding site, such as the active site or
a fibronectin domain of the RC Kinase polypeptide, can be derivatized to the wells
of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
[0143] Methods for detecting such complexes, in addition to those described above for the
GST-immobilized complexes, include immunodetection of complexes using antibodies which
specifically bind to the RC Kinase polypeptide (or polynucleotides) or test compound,
enzyme-linked assays which rely on detecting a RC Kinase activity of the RC Kinase
polypeptide, and SDS gel electrophoresis under non-reducing conditions.
[0144] Screening for test compounds which bind to a RC Kinase polypeptide or polynucleotide
also can be carried out in an intact cell. Any cell which comprises a RC Kinase polynucleotide
or polypeptide can be used in a cell-based assay system. A RC Kinase polynucleotide
can be naturally occurring in the cell or can be introduced using techniques such
as those described above. Either a primary culture or an established cell line, including
neoplastic cell lines such as the colon cancer cell lines HCT116, DLD1, HT29, Caco2,
SW837, SW480, and RKO, breast cancer cell lines 21-PT, 21-MT, MDA-468, SK-BR3, and
BT-474, the A549 lung cancer cell line, and the H392 glioblastoma cell line, can be
used. An intact cell is contacted with a test compound. Binding of the test compound
to a RC Kinase polypeptide or polynucleotide is determined as described above, after
lysing the cell to release the RC Kinase polypeptide-test compound complex.
Enzyme Assays
[0145] Test compounds can be tested for the ability to increase or decrease a RC Kinase
activity of a RC Kinase polypeptide. RC Kinase activity can be measured, for example,
using the methods referenced in Example 1. RC Kinase activity can be measured after
contacting either a purified RC Kinase polypeptide, a cell extract, or an intact cell
with a test compound. A test compound which decreases RC Kinase activity by at least
about 10, preferably about 50, more preferably about 75, 90, or 100% is identified
as a potential agent for decreasing signaling through a MAP kinase phosphorelay system,
production of inflammatory mediators, or extracellular matrix degradation. A test
compound which increases RC Kinase activity by at least about 10, preferably about
50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing
signaling through a MAP kinase phosphorelay system, production of inflammatory mediators,
or extracellular matrix degradation.
Gene Expression
[0146] In another embodiment, test compounds which increase or decrease RC Kinase gene expression
are identified. A RC Kinase polynucleotide is contacted with a test compound, and
the expression of an RNA or polypeptide product of the RC Kinase polynucleotide is
determined. The level of expression of RC Kinase mRNA or polypeptide in the presence
of the test compound is compared to the level of expression of RC Kinase mRNA or polypeptide
in the absence of the test compound. The test compound can then be identified as a
modulator of expression based on this comparison. For example, when expression of
RC Kinase mRNA or polypeptide is greater in the presence of the test compound than
in its absence, the test compound is identified as a stimulator or enhancer of RC
Kinase mRNA or polypeptide is less expression. Alternatively, when expression of the
mRNA or protein is less in the presence of the test compound than in its absence,
the test compound is identified as an inhibitor of RC Kinase mRNA or polypeptide expression.
[0147] The level of RC Kinase mRNA or polypeptide expression in the cells can be determined
by methods well known in the art for detecting mRNA or protein. Either qualitative
or quantitative methods can be used. The presence of polypeptide products of a RC
Kinase polynucleotide can be determined, for example, using a variety of techniques
known in the art, including immunochemical methods such as radioimmunoassay, Western
blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined
in
vivo, in a cell culture, or in an
in vitro translation system by detecting incorporation of labeled amino acids into a RC Kinase
polypeptide.
[0148] Such screening can be carried out either in a cell-free assay system or in an intact
cell. Any cell which expresses a RC Kinase polynucleotide can be used in a cell-based
assay system The RC Kinase polynucleotide can be naturally occurring in the cell or
can be introduced using techniques such as those described above. Either a primary
culture or an established cell line, including neoplastic cell lines such as the colon
cancer cell lines HGT116, DLD1, HT29, Caco2, SW837, SW480, and RKO, breast cancer
cell lines 21-PT, 21-M, MDA-468, SK-BR3, and BT-474, the A549 lung cancer cell line,
and the H392 glioblastoma cell line, can be used.
Pharmaceutical Compositions
[0149] The RC Kinase polypeptides of the present invention are suitable for use in pharmaceutical
compositions which can be administered to a patient to achieve a therapeutic effect.
Pharmaceutical compositions disclosed herein can comprise a RC Kinase polypeptide,
RC Kinase polynucleotide, antibodies which specifically bind to a RC Kinase polypeptide,
or mimetics, agonists, antagonists, or inhibitors of a RC Kinase polypeptide. The
compositions can be administered alone or in combination with at least one other agent,
such as stabilizing compound, which can be administered in any sterile, biocompatible
pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose,
and water. The compositions can be administered to a patient alone, or in combination
with other agents, drugs or hormones.
[0150] In addition to the active ingredients, these pharmaceutical compositions can contain
suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries
which facilitate processing of the active compounds into preparations which can be
used pharmaceutically. Pharmaceutical compositions disclosed herein can be administered
by any number of routes including, but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical
compositions for oral administration can be formulated using pharmaceutically acceptable
carriers well known in the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion
by the patient.
[0151] Pharmaceutical preparations for oral use can be obtained through combination of active
compounds with solid excipient, optionally grinding a resulting mixture, and processing
the mixture of granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers,
such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn,
wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylinethyl-cellulose,
or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins
such as gelatin and collagen. If desired, disintegrating or solubilizing agents can
be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a
salt thereof, such as sodium alginate.
[0152] Dragee cores can be used in conjunction with suitable coatings, such as concentrated
sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol
gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets
or dragee coatings for product identification or to characterize the quantity of active
compound,
i.
e., dosage.
[0153] Pharmaceutical preparations which can be used orally include push-fit capsules made
of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as
glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with
a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium
stearate, and, optionally, stabilizers. In soft capsules, the active compounds can
be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0154] Pharmaceutical formulations suitable for parenteral administration can be formulated
in aqueous solutions, preferably in physiologically compatible buffers such as Hanks'
solution, Ringer's solution, or physiologically buffered saline. Aqueous injection
suspensions can contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions
of the active compounds can be prepared as appropriate oily injection suspensions.
Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the
suspension also can contain suitable stabilizers or agents which increase the solubility
of the compounds to allow for the preparation of highly concentrated solutions. For
topical or nasal administration, penetrants appropriate to the particular barrier
to be permeated are used in the formulation. Such penetrants are generally known in
the art.
[0155] The pharmaceutical compositions disclosed herein can be manufactured in a manner
that is known in the art,
e.
g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical
composition can be provided as a salt and can be formed with many acids, including
but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic solvents than are
the corresponding free base forms. In other cases, the preferred preparation can be
a lyophilized powder which can contain any or all of the following: 1-50 mM histidine,
0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined
with buffer prior to use.
[0156] Further details on techniques for formulation and administration can be found in
the latest edition of
REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an
appropriate container and labeled for treatment of an indicated condition. Such labeling
would include amount, frequency, and method of administration.
Predictive, Diagnostic and Prognostic Assays
[0157] The present invention provides methods for determining whether a subject is at risk
for developing COPD and other disorders by detecting the disclosed biomarkers, i.e.,
the disclosed polynucleotide marker comprising the polynucleotide sequence of the
SEQ ID NO: 1, 2, 3, 4, 5, or 6 and/or the polypeptide markers encoded thereby or polypeptide
markers comprising the polypeptide sequences of the SEQ ID NO: 7,8,9,10,11, or 12.
[0158] In clinical applications, biological samples can be screened for the presence and/or
absence of the biomarkers identified herein. Such samples are for example needle biopsy
cores, surgical resection samples, or body fluids like serum, thin needle nipple aspirates
and urine. For example, these methods include obtaining a biopsy, which is optionally
fractionated by cryostat sectioning to enrich diseases cells to about 80% of the total
cell population. In certain embodiments, polynucleotides extracted from these samples
may be amplified using techniques well known in the art. The expression levels of
selected markers detected would be compared with statistically valid groups of diseased
and healthy samples.
[0159] In one embodiment the
in vitro diagnostic method comprises determining whether a subject has an abnormal mRNA and/or
protein level of the disclosed markers, such as by Northern blot analysis, reverse
transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation,
Western blot hybridization, or immunohistochemistry. According to the method, cells
are obtained from a subject and the levels of the disclosed biomarkers, protein or
mRNA level, is determined and compared to the level of these markers in a healthy
subject. An abnormal level of the biomarker polypeptide or mRNA levels is likely to
be indicative of diseases such as COPD.
[0160] In another embodiment the
in vitro diagnostic method comprises determining whether a subject has an abnormal DNA content
of said genes or said genomic loci, such as by Southern blot analysis, dot blot analysis,
Fluorescence or Colorimetric In Situ Hybridization, Comparative Genomic Hybridization
or quantitative PCR. In general these assays comprise the usage of probes from representative
genomic regions. The probes contain at least parts of said genomic regions or sequences
complementary or analogous to said regions. In particular intra- or intergenic regions
of said genes or genomic regions. The probes can consist of nucleotide sequences or
sequences of analogous functions (e.g. PNAs, Morpholino oligomers) being able to bind
to target regions by hybridization. In general genomic regions being altered in said
patient samples are compared with unaffected control samples (normal tissue from the
same or different patients, surrounding unaffected tissue, peripheral blood) or with
genomic regions of the same sample that don't have said alterations and can therefore
serve as internal controls. In a preferred embodiment regions located on the same
chromosome are used. Alternatively, gonosomal regions and /or regions with defined
varying amount in the sample are used. In one favored embodiment the DNA content,
structure, composition or modification is compared that lie within distinct genomic
regions. Especially favored are methods that detect the DNA content of said samples,
where the amount of target regions are altered by amplification and or deletions.
In another embodiment the target regions are analyzed for the presence of polymorphisms
(e.g. Single Nucleotide Polymorphisms or mutations) that affect or predispose the
cells in said samples with regard to clinical aspects, being of diagnostic, prognostic
or therapeutic value. Preferably, the identification of sequence variations is used
to define haplotypes that result in characteristic behavior of said samples with said
clinical aspects.
[0161] One embodiment of the invention is an
in vitro method for the prediction, diagnosis or prognosis of COPD by the detection of at
least 10, at least 5, or at least 4, or at least 3 and more preferably at least 2
markers whereby the markers are genes and/or genomic nucleic acid sequences that are
located on one chromosomal region which is altered in COPD.
[0162] The invention relates to an
in vitro method for the prediction, diagnosis or prognosis of respiratory diseases comprising
detecting at least one marker selected from the group consisting of: (a) a polynucleotide
of the invention; and (b) a RC Kinase polypeptide of the invention.
[0163] The invention also relates to the method as described above wherein the disease is
chronic obstructive pulmonary disease, cancer, or a respiratory disease in which cell
signaling is defective, and/or wherein the detection method comprises the use of PCR,
arrays or beads.
[0164] The invention also relates to an
in vitro method for the prediction, diagnosis or prognosis of COPD comprising detecting at
least one marker selected from the group consisting of: (a) a polynucleotide of the
invention; and (b) a RC Kinase polypeptide of the invention.
[0165] The invention also encompasses a method as described above, wherein the expression
level of the marker is detected.
[0166] One further embodiment of the invention is an
in vitro method for the prediction, diagnosis or prognosis of COPD by the detection of RC
Kinase gene and/or genomic nucleic acid sequence.
[0167] In one embodiment, the
in vitro method for the prediction, diagnosis or prognosis of COPD and COPD in particular
is done by the detection of:
- (a) polynucleotide of the SEQ ID NO: 1, 2, 3, 4, 5, or 6;
- (b) a polynucleotide which hybridizes under stringent conditions to a polynucleotide
specified in (a) encoding a polypeptide exhibiting the same biological function as
RC Kinase;
- (c) a polynucleotide the sequence of which deviates from the polynucleotide specified
in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting
the same biological function as RC Kinase; or
- (d) a polynucleotide which represents a specific fragment, derivative or allelic variation
of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting
the same biological function as RC Kinase;
in a biological sample comprising the following steps: hybridizing any polynucleotide
or analogous oligomer specified in (a) to (d) to a polynucleotide material of a biological
sample, thereby forming a hybridization complex; and detecting said hybridization
complex.
[0168] In another embodiment the
in vitro method for the prediction, diagnosis or prognosis of COPD is done as just described
but, wherein before hybridization, the polynucleotide material of the biological sample
is amplified.
[0169] In another embodiment the
in vitro method for the diagnosis or prognosis of COPD in particular is done by the detection
of:
- (a) a polynucleotide selected from the polynucleotides of the SEQ ID NO: 1, 2, 3,4,5,
or 6;
- (b) a polynucleotide which hybridizes under stringent conditions to a polynucleotide
specified in (a) encoding a polypeptide exhibiting the same biological function as
RC Kinase;
- (c) a polynucleotide the sequence of which deviates from the polynucleotide specified
in (a) and (b) due to the degeneracy of the genetic code encoding a polypeptide exhibiting
the same biological function as RC Kinase;
- (d) a polynucleotide which represents a specific fragment, derivative or allelic variation
of a polynucleotide sequence specified in (a) to (c) encoding a polypeptide exhibiting
the same biological function as RC Kinase;
- (e) a polypeptide encoded by a polynucleotide sequence specified in (a) to (d); or
- (f) a polypeptide comprising the polypeptide of SEQ ID NO: 7, 8, 9, 10, 11, or 12:
comprising the steps of contacting a biological sample with a reagent which specifically
interacts with the polynucleotide specified in (a) to (d) or the polypeptide specified
in (e) or (f).
1. DNA array technology
[0170] In one embodiment, the present Invention also provides a method wherein polynucleotide
probes are immobilized an a DNA chip in an organized array. Oligonucleotides can be
bound to a solid Support by a variety of processes, including lithography. For example
a chip can hold up to 41,000 oligonucleotides (GeneChip, Affymetrix). The present
invention provides significant advantages over the available tests for COPD, such
as COPD, because it increases the reliability of the test by providing an array of
polynucleotide markers on a single chip.
[0171] The method includes obtaining a biopsy of an affected person, which is optionally
fractionated by cryostat sectioning to enrich diseased cells to about 80% of the total
cell population and the use of body fluids such as serum or urine, serum or cell containing
liquids (e.g. derived from fine needle aspirates). The DNA or RNA is then extracted,
amplified, and analyzed with a DNA chip to determine the presence of absence of the
marker polynucleotide sequences. In one embodiment, the polynucleotide probes are
spotted onto a substrate in a two-dimensional matrix or array. samples of polynucleotides
can be labeled and then hybridized to the probes. Double-stranded polynucleotides,
comprising the labeled sample polynucleotides bound to probe polynucleotides, can
be detected once the unbound portion of the sample is washed away.
[0172] The probe polynucleotides can be spotted on substrates including glass, nitrocellulose,
etc. The probes can be bound to the Substrate by either covalent bonds or by non-specific
interactions, such as hydrophobic interactions. The sample polynucleotides can be
labeled using radioactive labels, fluorophores, chromophores, etc. Techniques for
constructing arrays and methods of using these arrays are described in
EP No. 0 799 897;
PCT No. WO 97/29212;
PCT No. WO 97/27317;
EP No. 0 785 280;
PCT No. WO 97/02357;
U.S. Pat. No. 5,593,839;
U.S. Pat. No. 5,578,832;
EP No. 0 728 520;
U.S. Pat. No. 5,599,695;
EP No. 0 721 016;
U.S. Pat. No. 5,556,752;
PCT No. WO 95/22058; and
U.S. Pat. No. 5,631,734. Further, arrays can be used to examine differential expression of genes and can
be used to determine gene function. For example, arrays of the instant polynucleotide
sequences can be used to determine if any of the polynucleotide sequences are differentially
expressed between normal cells and diseased cells, for example. High expression of
a particular message in a diseased sample, which is not observed in a corresponding
normal sample, can indicate a COPD specific protein.
[0173] Disclosed herein are probes and primers that are specific to the unique polynucleotide
markers disclosed herein.
[0174] In one embodiment, the method comprises using a polynucleotide probe to determine
the presence of malignant or COPD cells in particular in a tissue from a patient.
Specifically, the method comprises:
- 1) providing a polynucleotide probe comprising a nucleotide sequence at least 12 nucleotides
in length, preferably at least 15 nucleotides, more preferably, 25 nucleotides, and
most preferably at least 40 nucleotides, and up to all or nearly all of the coding
sequence which is complementary to a portion of the coding sequence of a polynucleotide
of the SEQ ID NO: 1, 2, 3, 4, 5, or 6 or a sequence complementary thereto and is
- 2) differentially expressed in COPD;
- 3) obtaining a tissue sample from a patient with COPD;
- 4) providing a second tissue sample from a patient with no COPD;
- 5) contacting the polynucleotide probe under stringent conditions with RNA of each
of said first and second tissue samples (e.g., in a Northern blot or in situ hybridization
assay); and
- 6) comparing (a) the amount of hybridization of the probe with RNA of the first tissue
sample, with (b) the amount of hybridization of the probe with RNA of the second tissue
sample;
wherein a statistically significant difference in the amount of hybridization with
the RNA of the first tissue sample as compared to the amount of hybridization with
the RNA of the second tissue sample is indicative of COPD and COPD in particular in
the first tissue sample.
2. Data analysis methods
[0175] Comparison of the expression levels of one or more "RC Kinase " with reference expression
levels, e.g., expression levels in diseased cells of COPD or in normal counterpart
cells, is preferably conducted using computer systems. Expression levels can be obtained
in two cells and these two sets of expression levels are introduced into a computer
system for comparison. One set of expression levels can be entered into a computer
system for comparison with values that are already present in the computer system,
or in computer-readable form that is then entered into the computer system.
[0176] Disclosed herein is a computer readable form of the gene expression profile data
of the invention, or of values corresponding to the level of expression of at least
one "RC Kinase" in a diseased cell. The values can be mRNA expression levels obtained
from experiments, e.g., microarray analysis. The values can also be mRNA levels normalized
relative to a reference gene whose expression is constant in numerous cells under
numerous conditions, e.g., GAPDH. The values in the computer can be ratios of, or
differences between, normalized or non-normalized mRNA levels in different samples.
[0177] The gene expression profile data can be in the form of a table, such as an Excel
table. The data can be alone, or it can be part of a larger database, e.g., comprising
other expression profiles. For example, the expression profile data of the invention
can be part of a public database. The computer readable form can be in a computer.
Disclosed herein is also a computer displaying the gene expression profile data.
[0178] Disclosed herein is a method for determining the similarity between the level of
expression of one or more "RC Kinase " in a first cell, e.g., a cell of a subject,
and that in a second cell, comprising obtaining the level of expression of one or
more "RC Kinase " in a first cell and entering these values into a computer comprising
a database including records comprising values corresponding to levels of expression
of one or more "RC Kinase" in a second cell, and processor instructions, e.g., a user
interface, capable of receiving a selection of one or more values for comparison purposes
with data that is stored in the computer. The computer may further comprise a means
for converting the comparison data into a diagram or chart or other type of output.
[0179] Values representing expression levels of "RC Kinase" can be entered into a computer
system, comprising one or more databases with reference expression levels obtained
from more than one cell. For example, the computer comprises expression data of diseased
and normal cells. Instructions are provided to the computer, and the computer is capable
of comparing the data entered with the data in the computer to determine whether the
data entered is more similar to that of a normal cell or of a diseased cell.
[0180] Disclosed herein is a computer that comprises values of expression levels in cells
of subjects at different stages of COPD, and the computer is capable of comparing
expression data entered into the computer with the data stored, and produce results
indicating to which of the expression profiles in the computer, the one entered is
most similar, such as to determine the stage of COPD in the subject.
[0181] The reference expression profiles in the computer can be expression profiles from
cells of COPD of one or more subjects, which cells are treated in
vivo or
in vitro with a drug used for therapy of COPD. Upon entering of expression data of a cell
of a subject treated
in vitro or in
vivo with the drug, the computer is instructed to compare the data entered to the data
in the computer, and to provide results indicating whether the expression data input
into the computer are more similar to those of a cell of a subject that is responsive
to the drug or more similar to those of a cell of a subject that is not responsive
to the drug. Thus, the results indicate whether the subject is likely to respond to
the treatment with the drug or unlikely to respond to it.
[0182] Disclosed herein is a system that comprises a means for receiving gene expression
data for one or a plurality of genes; a means for comparing the gene expression data
from each of said one or plurality of genes to a common reference frame; and a means
for presenting the results of the comparison. This system may further comprise a means
for clustering the data.
[0183] Disclosed herein is also a computer program for analyzing gene expression data comprising
(i) a computer code that receives as input gene expression data for a plurality of
genes and (ii) a computer code that compares said gene expression data from each of
said plurality of genes to a common reference frame.
[0184] Disclosed herein is also a machine-readable or computer-readable medium including
program instructions for performing the following steps: (i) comparing a plurality
of values corresponding to expression levels of one or more genes characteristic of
COPD in a query cell with a database including records comprising reference expression
or expression profile data of one or more reference cells and an annotation of the
type of cell; and (ii) indicating to which cell the query cell is most similar based
on similarities of expression profiles. The reference cells can be cells from subjects
at different stages of COPD. The reference cells can also be cells from subjects responding
or not responding to a particular drug treatment and optionally incubated
in vitro or in
vivo with the drug.
[0185] The reference cells may also be cells from subjects responding or not responding
to several different treatments. and the computer system indicates a preferred treatment
for the subject. Accordingly, disclosed herein is a method for selecting a therapy
for a patient having COPD, the method comprising: (i) providing the level of expression
of one or more genes characteristic of COPD in a diseased cell of the patient; (ii)
providing a plurality of reference profiles, each associated with a therapy, wherein
the subject expression profile and each reference profile has a plurality of values,
each value representing the level of expression of a gene characteristic of COPD;
and (iii) selecting the reference profile most similar to the subject expression profile,
to thereby select a therapy for said patient. Step (iii) can be performed by a computer.
The most similar reference profile may be selected by weighing a comparison value
of the plurality using a weight value associated with the corresponding expression
data.
[0186] The relative abundance of an mRNA in two biological samples can be scored as a perturbation
and its magnitude determined (i.e., the abundance is different in the two sources
of mRNA tested), or as not perturbed (i.e., the relative abundance is the same). A
difference between the two sources of RNA of at least a factor of about 25% (RNA from
one source is 25% more abundant in one source than the other source), more usually
about 50%, even more often by a factor of about 2 (twice as abundant), 3 (three times
as abundant) or 5 (five times as abundant) can be scored as a perturbation. Perturbations
can be used by a computer for calculating and expression comparisons.
[0187] Preferably, in addition to identifying a perturbation as positive or negative, it
is advantageous to determine the magnitude of the perturbation. This can be carried
out, as noted above, by calculating the ratio of the emission of the two fluorophores
used for differential labeling, or by analogous methods that will be readily apparent
to those of skill in the art.
[0188] The computer readable medium may further comprise a pointer to a descriptor of a
stage of COPD or to a treatment for COPD.
[0189] In operation, the means for receiving gene expression data, the means for comparing
the gene expression data, the means for presenting, the means for normalizing, and
the means for clustering within the context of the systems disclosed herein can involve
a programmed computer with the respective functionalities described herein, implemented
in hardware or hardware and software; a logic circuit or other component of a programmed
computer that performs the operations specifically identified herein, dictated by
a computer program; or a computer memory encoded with executable instructions representing
a computer program that can cause a computer to function in the particular fashion
described herein.
[0190] Those skilled in the art will understand that the systems and methods disclosed herein
may be applied to a variety of systems, including IBM-compatible personal computers
running MS-DOS or Microsoft Windows.
[0191] The computer may have internal components linked to external components. The internal
components may include a processor element interconnected with a main memory. The
computer system can be an Intel Pentium
®-based processor of 200 MHz or greater clock rate and with 32 MB or more of main memory.
The external component may comprise a mass storage, which can be one or more hard
disks (which are typically packaged together with the processor and memory). Such
hard disks are typically of 1 GB or greater storage capacity. Other external components
include a user interface device, which can be a monitor, together with an inputting
device, which can be a "mouse", or other graphic input devices, and/or a keyboard.
A printing device can also be attached to the computer.
[0192] Typically, the computer system is also linked to a network link, which can be part
of an Ethernet link to other local computer systems, remote computer systems, or wide
area communication networks, such as the Internet. This network link allows the computer
system to share data and processing tasks with other computer systems.
[0193] Loaded into memory during operation of this system are several software components,
which are both standard in the art and special to the instant disclosure. These software
components collectively cause the computer system to function according to the methods
disclosed herein These software components are typically stored on a mass storage.
A software component represents the operating system, which is responsible for managing
the computer system and its network interconnections. This operating system can be,
for example, of the Microsoft Windows' family, such as Windows 95, Windows 98, Windows
NT or Windows XP. A software component represents common languages and functions conveniently
present on this system to assist programs implementing the methods specific to this
disclosure. Many high or low level computer languages can be used to program the analytic
methods disclosed herein. Instructions can be interpreted during run-time or compiled.
Preferred languages include C/C++, and JAVA
®. Most preferably, the methods disclosed herein are programmed in mathematical software
packages which allow symbolic entry of equations and high-level specification of processing,
including algorithms to be used, thereby freeing a user of the need to procedurally
program individual equations or algorithms. Such packages include Matlab from Mathworks
(Natick, Mass.), Mathematica from Wolfram Research (Champaign, Ill.), or S-Plus from
Math Soft (Cambridge, Mass.). Accordingly, a software component represents the analytic
methods disclosed herein as programmed in a procedural language or symbolic package.
The compute system can also contain a database comprising values representing levels
of expression of one or more genes characteristic of COPD. The database may contain
one or more expression profiles of genes characteristic of COPD in different cells.
[0194] In an exemplary implementation, to practice the methods disclosed herein a user first
loads expression profile data into the computer system. These data can be directly
entered by the user from a monitor and keyboard, or from other computer systems linked
by a network connection, or on removable storage media such as a CD-ROM or floppy
disk or through the network. Next the user causes execution of expression profile
analysis software which performs the steps of comparing and, e.g., clustering co-varying
genes into groups of genes.
[0195] In another exemplary implementation, expression profiles are compared using a method
described in
U.S. Patent No. 6,203,987. A user first loads expression profile data into the computer system. Creneset profile
definitions are loaded into the memory from the storage media or from a remote computer,
preferably from a dynamic geneset database system, through the network. Next the user
causes execution of projection, software which performs the steps of converting expression
profile to projected expression profiles. The projected expression profiles are then
displayed.
[0196] In yet another exemplary implementation, a user first leads a projected profile into
the memory. The user then causes the loading of a reference profile into the memory.
Next, the user causes the execution of comparison software which performs the steps
of objectively comparing the profiles.
3. Detection of variant polynucleotide sequence
[0197] Disclosed herein are methods for determining whether a subject is at risk for developing
a disease, such as a predisposition to develop COPD, for example COPD, associated
with an aberrant activity of any one of the polypeptides encoded by any of the polynucleotides
of the SEQ ID NO: 1, wherein the aberrant activity of the polypeptide is characterized
by detecting the presence or absence of a genetic lesion characterized by at least
one of these:
- (i) an alteration affecting the integrity of a gene encoding a marker polypeptides,
or
- (ii) the misexpression of the encoding polynucleotide.
[0198] To illustrate, such genetic lesions can be detected by ascertaining the existence
of at least one of these:
- I. a deletion of one or more nucleotides from the polynucleotide sequence
- II. an addition of one or more nucleotides to the polynucleotide sequence
- III. a substitution of one or more nucleotides of the polynucleotide sequence
- IV. a gross chromosomal rearrangement of the polynucleotide sequence
- V. a gross alteration in the level of a messenger RNA transcript of the polynucleotide
sequence
- VI. aberrant modification of the polynucleotide sequence, such as of the methylation
pattern of the genomic DNA
- VII. the presence of a non-wild type splicing pattern of a messenger RNA transcript
of the gene
- VIII. a non-wild type level of the marker polypeptide
- IX. allelic loss of the gene
- X. inappropriate post-translational modification of the marker polypeptide
[0199] Disclosed herein are assay techniques for detecting mutations in the encoding polynucleotide
sequence. These methods include, but are not limited to, methods involving sequence
analysis, Southern blot hybridization, restriction enzyme site mapping, and methods
involving detection of absence of nucleotide pairing between the polynucleotide to
be analyzed and a probe.
[0200] Specific diseases or disorders, e.g., genetic diseases or disorders, are associated
with specific allelic variants of polymorphic regions of certain genes, which do not
necessarily encode a mutated protein. Thus, the presence of a specific allelic variant
of a polymorphic region of a gene in a subject can render the subject susceptible
to developing a specific disease or disorder. Polymorphic regions in genes, can be
identified, by determining the nucleotide sequence of genes in populations of individuals.
If a polymorphic region is identified, then the link with a specific disease can be
determined by studying specific populations of individuals, e.g. individuals which
developed a specific disease, such as COPD. A polymorphic region can be located in
any region of a gene, e.g., exons, in coding or non coding regions of exons, introns,
and promoter region.
[0201] A polynucleotide composition can comprise a polynucleotide probe including a region
of nucleotide sequence which is capable of hybridising to a sense or antisense sequence
of a gene or naturally occurring mutants thereof, or 5' or 3' flanking sequences or
intronic sequences naturally associated with the subject genes or naturally occurring
mutants thereof. The polynucleotide of a cell is rendered accessible for hybridization,
the probe is contacted with the polynucleotide of the sample, and the hybridization
of the probe to the sample polynucleotide is detected. Such techniques can be used
to detect lesions or allelic variants at either the genomic or mRNA level, including
deletions, substitutions, etc., as well as to determine mRNA transcript levels.
[0202] A preferred detection method is allele specific hybridization using probes overlapping
the mutation or polymorphic site and having about 5, 10, 20, 25, or 30 nucleotides
around the mutation or polymorphic region. Several probes capable of hybridising specifically
to allelic variants can be attached to a solid phase support, e.g., a "chip". Mutation
detection analysis using these chips comprising oligonucleotides, also termed "
DNA probe arrays" is descibed e.g., in Cronin et al. (119). A chip can comprise all the allelic variants of at least one polymorphic region
of a gene. The solid phase support is then contacted with a test polynucleotide and
hybridization to the specific probes is detected. Accordingly, the identity of numerous
allelic variants of one or more genes can be identified in a simple hybridization
experiment.
[0203] Detection of the lesion can comprise utilizing the probe/primer in a polymerase chain
reaction (PCR) (see, e.g.
U.S. Patent Nos. 4,683,195 and
4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligase chain reaction (LCR)
[Landegran et al., 1988, (120) and Nakazawa et al., 1994 (121)], the latter of which
can be particularly useful for detecting point mutations in the gene; Abravaya et
al., 1995 ,(122)]. In a merely illustrative embodiment, the method includes the steps
of (i) collecting a sample of cells from a patient, (ii) isolating polynucleotide
(e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the polynucleotide
sample with one or more primers which specifically hybridize to a polynucleotide sequence
under conditions such that hybridization and amplification of the polynucleotide (if
present) occurs, and (iv) detecting the presence or absence of an amplification product,
or detecting the size of the amplification product and comparing the length to a control
sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used for detecting mutations
described herein.
[0204] Alternative amplification methods include: self sustained sequence replication [Guatelli,
J.C. et al., 1990, (123)], transcriptional amplification system [Kwoh, D.Y. et al.,
1989, (124)], Q-Beta replicase [Lizardi, P.M. et al., 1988 ,(125)], or any other polynucleotide
amplification method, followed by the detection of the amplified molecules using techniques
well known to those of skill in the art. These detection schemes are especially useful
for the detection of polynucleotide molecules if such molecules are present in very
low numbers.
[0205] In the subject assay, mutations in, or allelic variants, of a gene from a sample
cell can be identified by alterations in restriction enzyme cleavage patterns. For
example, sample and control DNA is isolated, amplified (optionally), digested with
one or more restriction endonucleases, and fragment length sizes are determined by
gel electrophoresis. Moreover; the use of sequence specific ribozymes (see, for example,
U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss
of a ribozyme cleavage site.
4. In situ hybridization
[0206] In one aspect, the method comprises
in situ hybridization with a probe derived from a given marker polynucleotide, which sequence
is selected from any of the polynucleotide sequences of the SEQ ID NO: 1, 2, 3, 4,
5, or 6 or a sequence complementary thereto. The method comprises contacting the labeled
hybridization probe with a sample of a given type of tissue from a patient potentially
having COPD and COPD in particular as well as normal tissue from a person with no
COPD, and determining whether the probe labels tissue of the patient to a degree significantly
different (e.g., by at least a factor of two, or at least a factor of five, or at
least a factor of twenty, or at least a factor of fifty) than the degree to which
normal tissue is labeled.
Therapeutic Indications and Methods
[0207] The human RC Kinase disclosed herein is likely to be useful for the same purposes
as previously identified serine/threonine kinases, or more specifically for the same
purposes as previously identified MAPK kinase kinases. For example, transforming growth
factor type beta (TGF-β) regulates the proliferation and differentiation of a variety
of cell types binding to and activating cell surface receptors which possess serine/threonine
kinase activity.
Atfi et al. (Proc. Natl. Acad. Sci. U.S.A. 92, 12110-04, 1995) have shown that TGF-β activates a 78-kDa protein (p78) serine/threonine kinase;
the p78 kinase was activated only in cells for which TGF-β acts as a growth inhibitory
factor. Another example is MAPKKK3, the human kinase to which RC Kinase is most closely
related. MAPKKK3 is known to directly regulate the stress-activated protein kinase
(SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by activating
SEK and MEK1/2 respectively. It can also enhance transcription from a nuclear factor
kappa-B (NFκB)-dependent reporter gene. Other previously identified MAPK kinase kinases
include c-Raf, Mos, MEKK1, B-Raf, TAK1, A-Raf, Tpl-2, MEKK2, MUK, SPRK, MAPKKK5, MEKK4,
and MST. The human RC Kinase disclosed herein also may be involved in such signaling.
Thus, regulation of its activity can be used to treat disorders in which such signaling
is defective or dysregulated.
[0208] Expression profiling of RC Kinase showed that it is expressed highly in the lung
and trachea, and that it can be found to be expressed in activated B cells and other
leukocytes. Some of the ESTs which are expressed from human RC Kinase, e.g., GenBank
accession numbers BU676900, BG484791, CA311871, BQ045211, and BM969829, are also expressed
in the lung epithelial cells and in primary lung cystic fibrosis epithelial cells.
Furthermore, microarray analysis comparing lungs from patients with chronic obstructive
pulmonary disease (COPD) and lungs from normal lungs showed that RC Kinase is upregulated
an average of 4.58 fold in the COPD lungs (figure 1). Thus, human RC Kinase could
be a potential target for treating lung disease, such as COPD.
[0209] COPD is a condition defined physiologically as airflow obstruction that generally
results from a mixture of emphysema and peripheral airway obstruction due to chronic
bronchitis (
Senior & Shapiro, Pulmonary Diseases and Disorders, 3d ed., New York, McGraw-Hill,
1998, pp. 659-681, 1998;
Barnes, Chest 117, 10S-14S, 2000). Emphysema is characterized by destruction of alveolar walls leading to abnormal
enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically
as the presence of chronic productive cough for three months in each of two successive
years. In COPD, airflow obstruction is usually progressive and is only partially reversible.
By far the most important risk factor for development of COPD is cigarette smoking,
although the disease does occur in non-smokers.
[0210] The expression of RC Kinase in cell increase significantly after treatment of the
cells with 95 mM potassium chloride (KCl), which subjects the cells to a hyperosmotic
stress. Additionally, some cell lines increase its expression of RC Kinase in response
to 500 µm hydrogen peroxide (H
2O
2), a treatment which subjects the cells to an oxidative stress and which has been
reported to impair the capacity of B cells to stimulate specific T cells. Such upregulation
of RC Kinase in the cell lines in response to hyperosmotic and oxidative stress suggests
that higher expression of RC Kinase in the lungs of COPD patients may be the result
of cellular stresses caused by the irritants in tobacco smoke or stresses caused by
the inflammatory response to those irritants.
[0211] Chronic inflammation of the airways is a key pathological feature of COPD (Senior
& Shapiro, 1998). The inflammatory cell population comprises increased numbers of
macrophages, neutrophils, and CD8
+ lymphocytes. Inhaled irritants, such as tobacco smoke, activate macrophages which
are resident in the respiratory tract, as well as epithelial cells leading to release
of chemokines (
e.
g., interleukin-8) and other chemotactic factors. These chemotactic factors act to
increase the trafficking of neutrophils, monocytes, and lymphocytes from the blood
into the lung tissue and airways. CD8
+ lymphocytes recruited into the airways can recognize proteins that have been altered
by inhaled chemicals, such as tobacco smoke, and induce apoptosis of the cells expressing
such altered proteins. The lymphocytes can also release inflammatory mediators to
recruit other leukocytes to the lungs. Apoptotic cells killed by the lymphocytes can
additionally release proteolytic enzymes. Neutrophils and monocytes recruited into
the airways can release a variety of potentially damaging mediators such as proteolytic
enzymes and reactive oxygen species. Although not as well studied, B cells have also
been found in increased numbers in the lungs of smokers with airway obstruction, particularly
within lymphoid nodules that develop in the airway adventitia (
Bosken, CH et al., Am Rev Respir Dis., 145(4 Pt 1):911-7, 1992). The B cells may play roles in antigen presentation, inflammatory cytokine production,
and the generation of antibodies such as IgE and IgA that target, promote, and maintain
the immune response in the lungs of COPD sufferers. Matrix degradation and emphysema,
along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion,
all are potential sequelae of this inflammatory response that lead to impaired airflow
and gas exchange.
[0212] RC Kinase has the ability to phosphorylate the MAP kinase kinase MKK4, and to a lesser
extent, the MAP kinase kinase MKK6, indicating that RC Kinase is an upstream activator
in one or more MAP kinase signaling cascades. As described above, the activation of
MAP kinase signaling cascades has many cellular consequences, including cell proliferation,
differentiation, adaptation to environmental stress, cytokine production, and apoptosis.
The activation of MKK4 has been shown in many cases to lead to the phosphorylation
of JNK-type MAP kinases, which in turn can activate c-Jun, a component of the AP-1
transcription factor complex. JNK-type MAP kinases are also known to inhibit NFAT
transcription factors. In addition, other MAPK kinase kinases, such as MEKK1, that
are initiators of the signaling cascades that result in the phosphorylation of JNK-type
MAP kinases, have been found to be able to activate the transcription factor NFκB,
indicating that this transcription factor is also a downstream target of these cascades.
Activation of MKK6, on the other hand, leads to the phosphorylation of p38-type MAP
kinases, which are known to be important in the activation of the immune response
and are key regulators of inflammatory cytokine expression.
[0213] RC Kinase appears to be upregulated and possibly activated by cellular stress, then
phosphorylates MKK4 (and to a lesser extent MKK6), which leads to the activation of
transcription factors AP-1 and NFkB. As a result of activation of this signaling cascade,
Interleukin-8 production is increased and leads to the recruitment of inflammatory
cells, such as neutrophils, that play a role in the pathology of COPD. The occurrence
of cellular stress, the activation of the transcription factors AP-1 and NF□B, and
the overproduction of Interleukin-8 are characteristic of numerous inflammatory diseases,
many of which may be treated or prevented through the regulation of RC Kinase. Such
diseases include asthma; allergic rhinitis; atopic dermatitis; hives; conjunctivitis;
vernal catarrh; chronic arthrorheumatism; systemic lupus erythematosus; psoriasis;
diabrotic colitis; systemic inflammatory response syndrome (SIRS); sepsis; polymyositis;
dermatomyositis (DM); Polyaritis nodoa (PN); mixed connective tissue disease (MCTD);
Sjoegren's syndrome; and gout.
[0214] Human RC Kinase ESTs, e.g., GenBank accession number BI832332, BX090530, N47620,
and N57475, also are expressed in normal brain medulla and in multiple sclerosis lesions
in the central nervous system. Thus, human RC Kinase could be a potential target for
treating multiple sclerosis and other central nervous system disorders.
[0215] Human RC Kinase ESTs, e.g., GenBank accession number AI68344-7, also are expressed
in well-differentiated endometrial adenocarcinoma. Thus, human RC Kinase could be
a potential target for treating cancers. Cancer is a disease fundamentally caused
by oncogenic cellular transformation. There are several hallmarks of transformed cells
that distinguish them from their normal counterparts and underlie the pathophysiology
of cancer. These include uncontrolled cellular proliferation, unresponsiveness to
normal death-inducing signals (immortalization), increased cellular motility and invasiveness,
increased ability to recruit blood supply through induction of new blood vessel formation
(angiogenesis), genetic instability, and dysregulated gene expression. Various combinations
of these aberrant physiologies, along with the acquisition of drug-resistance frequently
lead to an intractable disease state in which organ failure and patient death ultimately
ensue.
[0216] Most standard cancer therapies target cellular proliferation and rely on the differential
proliferative capacities between transformed and normal cells for their efficacy.
This approach is hindered by the facts that several important normal cell types are
also highly proliferative and that cancer cells frequently become resistant to these
agents. Thus, the therapeutic indices for traditional anti-cancer therapies rarely
exceed 2.0.
[0217] The advent of genomics-driven molecular target identification has opened up the possibility
of identifying new cancer-specific targets for therapeutic intervention that will
provide safer, more effective treatments for cancer patients. Thus, newly discovered
tumor-associated genes and their products can be tested for their role(s) in disease
and used as tools to discover and develop innovative therapies. Genes playing important
roles in any of the physiological processes outlined above can be characterized as
cancer targets.
[0218] Genes or gene fragments identified through genomics can readily be expressed in one
or more heterologous expression systems to produce functional recombinant proteins.
These proteins are characterized
in vitro for their biochemical properties and then used as tools in high-throughput molecular
screening programs to identify chemical modulators of their biochemical activities.
Agonists and/or antagonists of target protein activity can be identified in this manner
and subsequently tested in cellular and
in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative
testing in biological models and detailed pharmacokinetic and toxicological analyses
form the basis for drug development and subsequent testing in humans.
[0219] Disclosed herein is the use of novel agents identified by the screening assays described
above. Accordingly, disclosed herein is to use a test compound identified as described
herein in an appropriate animal model. For example, an agent identified as described
herein (
e.
g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme,
or a polypeptide-binding partner) can be used in an animal model to determine the
efficacy, toxicity, or side effects of treatment with such an agent. Alternatively,
an agent identified as described herein can be used in an animal model to determine
the mechanism of action of such an agent. Furthermore, this invention pertains to
uses of novel agents identified by the above-described screening assays for treatments
as described herein.
[0220] A reagent which affects RC Kinase activity can be administered to a human cell, either
in vitro or
in vivo, to reduce RC Kinase activity. The reagent preferably binds to an expression product
of a human RC Kinase gene. If the expression product is a polypeptide, the reagent
is preferably an antibody. For treatment of human cells
ex vivo, an antibody can be added to a preparation of stem cells which have been removed
from the body. The cells can then be replaced in the same or another human body, with
or without clonal propagation, as is known in the art.
[0221] The reagent can be delivered using a liposome. Preferably, the liposome is stable
in the animal into which it has been administered for at least about 30 minutes, more
preferably for at least about 1 hour, and even more preferably for at least about
24 hours. A liposome comprises a lipid composition that is capable of targeting a
reagent, particularly a polynucleotide, to a particular site in an animal, such as
a human. Preferably, the lipid composition of the liposome is capable of targeting
to a specific organ of an animal, such as the lung or liver.
[0222] A liposome useful in the present disclosure comprises a lipid composition that is
capable of fusing with the plasma membrane of the targeted cell to deliver its contents
to the cell. Preferably, the transfection efficiency of a liposome is about 0.5 µg
of DNA per 16 nmole of liposome delivered to about 10
6 cells, more preferably about 1.0 µg of DNA per 16 nmol of liposome delivered to about
10
6 cells, and even more preferably about 2.0 µg of DNA per 16 nmol of liposome delivered
to about 10
6 cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between
about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.
[0223] Suitable liposomes for use in the present disclosure include those liposomes standardly
used in, for example, gene delivery methods know to those of skill in the art. More
preferred liposomes include liposomes having a polycationic lipid composition and/or
liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally,
a liposome comprises a compound capable of targeting the liposome to a tumor cell,
such as a tumor cell ligand exposed on the outer surface of the liposome.
[0224] Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme
can be achieved using methods which are standard in the art (see, for example,
U.S. Patent 5,705,151). Preferably, from about 0.1 µg to about 10 µg of polynucleotide is combined with
about 8 nmol of liposomes, more preferably from about 0.5 µg to about 5 µg of polynucleotides
are combined with about 8 nmol liposomes, and even more preferably about 1.0 µg of
polynucleotides is combined with about 8 nmol liposomes.
[0225] Antibodies can be delivered to specific tissues
in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques
are taught in, for example,
Findeis et al. Trends in Biotechnol. 11, 202-05 (1993);
Chiou et al., GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER
(J.A. Wolff, ed.) (1994);
Wu & Wu, J. Biol. Chem. 263, 621-24 (1988);
Wu et al., J. Biol. Chem. 269, 542-46 (1994);
Zenke et al., Proc. Natl, Acad Sci. U.S.A. 87, 3655-59 (1990);
Wu et al., J. Biol. Chem. 266, 338-42 (1991).
[0226] If the reagent is a single-chain antibody, polynucleotides encoding the antibody
can be constructed and introduced into a cell either
ex vivo or
in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated
DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated
cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast
fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated
transfection.
Determination of a Therapeutically Effective Dose
[0227] The determination of a therapeutically effective dose is well within the capability
of those skilled in the art. A therapeutically effective dose refers to that amount
of active ingredient which increases or decreases extracellular matrix degradation
relative to that which occurs in the absence of the therapeutically effective dose.
[0228] For any compound, the therapeutically effective dose can be estimated initially either
in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs.
The animal model also can be used to determine the appropriate concentration range
and route of administration. Such information can then be used to determine useful
doses and routes for administration in humans.
[0229] Therapeutic efficacy and toxicity,
e.g., ED
50 (the dose therapeutically effective in 50% of the population) and LD
50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic
effects is the therapeutic index, and it can be expressed as the ratio, LD
50/ED
50.
[0230] Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies is used in formulating
a range of dosage for human use. The dosage contained in such compositions is preferably
within a range of circulating concentrations that include the ED
50 with little or no toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of administration.
[0231] The exact dosage will be determined by the practitioner, in light of factors related
to the subject that requires treatment. Dosage and administration are adjusted to
provide sufficient levels of the active ingredient or to maintain the desired effect.
Factors which can be taken into account include the severity of the disease state,
general health of the subject, age, weight, and gender of the subject, diet, time
and frequency of administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered
every 3 to 4 days, every week, or once every two weeks depending on the half-life
and clearance rate of the particular formulation.
[0232] Normal dosage amounts can vary from 0.1 to 100,000 µgrams, up to a total dose of
about 1 µg, depending upon the route of administration. Guidance as to particular
dosages and methods of delivery is provided in the literature and generally available
to practitioners in the art. Those skilled in the art will employ different formulations
for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides
or polypeptides will be specific to particular cells, conditions, locations, etc.
[0233] Effective
in vivo dosages of an antibody are in the range of about 5 µg to about 50 µg/kg, about 50
µg to about 5 mg/kg, about 100 µg to about 500 µg/kg of patient body weight, and about
200 to about 250 µg/kg of patient body weight. For administration of polynucleotides
encoding single-chain antibodies, effective
in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg,
about 1 µg to about 2 mg, about 5 µg to about 500 µg, and about 20 µg to about 100
µg of DNA.
[0234] If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide
or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes
can be introduced into cells by a variety of methods, as described above.
[0235] Preferably, a reagent reduces expression of a RC Kinase polynucleotide or activity
of a RC Kinase polypeptide by at least about 10, preferably about 50, more preferably
about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of
the mechanism chosen to decrease the level of expression of a RC Kinase polynucleotide
or the activity of a RC Kinase polypeptide can be assessed using methods well known
in the art, such as hybridization of nucleotide probes to RC Kinase-specific mRNA,
quantitative RT-PCR, immunologic detection of a RC Kinase polypeptide, or measurement
of RC Kinase activity.
[0236] Any of the pharmaceutical compositions disclosed herein can be administered in combination
with other appropriate therapeutic agents. Selection of the appropriate agents for
use in combination therapy can be made by one of ordinary skill in the art, according
to conventional pharmaceutical principles. The combination of therapeutic agents can
act synergistically to effect the treatment or prevention of the various disorders
described above. Using this approach, one may be able to achieve therapeutic efficacy
with lower dosages of each agent, thus reducing the potential for adverse side effects.
[0237] Any of the therapeutic methods described above can be applied to any subject in need
of such therapy, including, for example, mammals such as dogs, cats, cows, horses,
rabbits, monkeys, and most preferably, humans.
EXAMPLE 1
Detection of RC Kinase activity
[0238] For high level expression of a FLAG-tagged RC Kinase polypeptide, HEK293 cells were
transfected with the expression vector pcDNA3.1-RC Kinase polypeptide (expressing
the DNA-sequence of ID NO: 1 with a FLAG epitope sequence at its amino terminal) using
the transfection reagent Polyfect (Qiagen). The cells were harvested 48 h after infection
and lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X, 1 mM NaF, 1
mM Na
3VO
4, and Proteinase Inhibitor Cocktail (Roche). RC Kinase polypeptide was then immunoprecipitated
from the lysate using anti-FLAG antibodies. An in vitro kinase assay was performed
in a volume of 40 µl with immunoprecipitated FLAG-RC Kinase polypeptide in 50 mM Tris-HCl,
pH 8.0, 50 mM NaCl, 5 mM MgCl
2, 1 mM dithiothreitol. The reaction is started by the addition of 4 µl of 1 mM ATP
supplemented with 5 µCi of (-
32P)ATP and incubated for 30 min at 37°C. Afterward, the samples were subjected to SDS-PAGE
and phosphorylated proteins were detected by autoradiography. Histone H1, myelin basic
protein, MEK2, MKK4, and MKK6 were tested as substrates. It was shown that the polypeptide
with the amino acid sequence of SEQ ID NO: 7 has RC Kinase, activity, specifically
that it has the ability to phosphorylate other RC Kinase polypeptides, MKK4, and MKK6.(Fig.8)
EXAMPLE 2
Identification of a test compound which binds to a RC Kinase polypeptide
[0239] Purified RC Kinase polypeptides comprising a glutathione-S-transferase protein and
absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted
with test compounds from a small molecule library at pH 7.0 in a physiological buffer
solution. RC Kinase polypeptides comprise an amino acid sequence shown in SEQ ID NOS:
2, 5, 6, or 7. The test compounds comprise a fluorescent tag. The samples are incubated
for 5 minutes to one hour. Control samples are incubated in the absence of a test
compound.
[0240] The buffer solution containing the test compounds is washed from the wells. Binding
of a test compound to a RC Kinase polypeptide is detected by fluorescence measurements
of the contents of the wells. A test compound which increases the fluorescence in
a well by at least 15% relative to fluorescence of a well in which a test compound
was not incubated is identified as a compound which binds to a RC Kinase polypeptide.
EXAMPLE 3
Identification of a test compound which decreases RC Kinase activity
[0241] RC Kinase polypeptides, purified as described in Example 1, are contacted with test
compounds from a small molecule library and assayed for RC Kinase activity using any
of the substrates mentioned in Example 1 or other substrates of RC Kinase. As controls,
RC Kinase polypeptides in the absence of a test compound also are assayed. Kinase
activity is measured as described in Example 1 or as taught in
Trost et al., J. Biol. Chem. 275, 7373-77, 2000;
Hayashi et al., Biochem. Biophys. Res. Commun. 264, 449-56, 1999;
Masure et al., Eur. J. Biochem. 265, 353-60, 1999; and
Mukhopadhyay et al., J. Bacteriol. 181, 6615-22, 1999.
[0242] Alternatively, RC Kinase activity can be measured indirectly by measuring downstream
effects, for example, by assaying NFκB or AP-1 reporter gene activity in RC Kinase
expressing cells, or by assaying Interleukin-8 production by RC kinase expressing
cells. For example, MercuryTM Pathway Profiling Luciferase System 2(CLONTECH) reporter
genes can be used by transfecting cells as well as RC Kinase expression vector. Since
in the presence of AP-1 or NFκB reporter as well as RC Kinase in transfected cell
show strong Luciferase activity, these cells can monitor the efficacy of a compound
whether it inhibit RC Kinase or not by measuring its reporter activity indirectly.
RC Kinase expressing cells are contacted with test compounds from a small molecule
library and assayed for Interleukin-8 production is also able to monitor RC Kinase
activity indirectly. As controls, the same assays are conducted in the absence of
test compounds.
[0243] A test compound which decreases RC Kinase activity relative to the control by at
least 20% is identified as a RC Kinase inhibitor.
EXAMPLE 4
Identification of a test compound which decreases RC Kinase gene expression
[0244] A test compound is administered to a culture of cells transfected with an expression
construct which expresses RC Kinase and incubated at 37°C for 10 to 45 minutes. A
culture of the same type of cells incubated for the same time without the test compound
provides a negative control.
[0245] RNA is isolated from the two cultures as described in
Chirgwin et al., Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 µg total RNA and hybridized with a
32P-labeled RC Kinase-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises
at least 11 contiguous nucleotides selected from the complement of SEQ ID NO: 1, 2,
3, 4, 5, or 6. A test compound which decreases the RC Kinase -specific signal relative
to the signal obtained in the absence of the test compound is identified as an inhibitor
of RC Kinase gene expression.
EXAMPLE 5
Treatment of chronic obstructive pulmonary disease with a reagent which specifically
binds to a RC Kinase gene product
[0246] Synthesis of antisense RC Kinase oligonucleotides comprising at least 11 contiguous
nucleotides selected from the complement of SEQ ID NO: 1, 2, 3, 4, 5, or 6 is performed
on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure
(
Uhlmann et al., Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol-precipitated
twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration.
Purity of these oligonucleotides is tested by capillary gel electrophoreses and ion
exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined
using the
Limulus Amebocyte Assay (
Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).
[0247] An aqueous composition containing the antisense oligonucleotides at a concentration
of 0.1-100 µM is administered intrabronchially to a patient with chronic obstructive
pulmonary disease. The severity of the disease is suppressed due to decreased RC Kinase
activity.
EXAMPLE 6
Disease-specific expression of RC Kinase detected by microarray analysis
Target preparation
[0248] Human total RNA was prepared from frozen lung tissue obtained from four normal individuals
and three individuals diagnosed with chronic obstructive pulmonary disease (Analytical
Biological Services Inc. Wilmington, DE, USA) using Trizol
™ (Invitrogen Corp., Carlsbad, CA, USA). Five micrograms of each of the total RNAs
was added to a reaction mix in a final volume of 12 µl, containing bacterial control
mRNAs (2.5 pg/µl araB/entF, 8.33 pg/µl fixB/gnd and 25 pg/µl hisB/leuB) and 1.0 µl
of 0.5 pmol/µl T7-(dT)
24 oligonucleotide primer. The mixture was incubated for 10 min at 70°C and chilled
on ice. With the mixture remaining on ice, 4 µl of 5x first-strand buffer, 2 µl 0.1
M DTT, 1 µl of 10 mM dNTP mix and 1 µl Superscript
™ II RNase H- reverse transcriptase (200 U/µl) was added to make a final volume of
20 µl, and the mixture incubated for 1 h in a 42°C water bath. Second-strand cDNA
was synthesized in a final volume of 150 µl, in a mixture containing 30 µl of 5x second-strand
buffer, 3 µl of 10 mM dNTP mix, 4 µl of
Escherichia coli DNA polymerase I (10 U/µl) and 1 µl of RNase H (2 U/µl) for 2 h at 16°C. The cDNA
was purified using a Qiagen QIAquick purification kit, dried down, and resuspended
in IVT reaction mix, containing 3.0 µl nuclease-free water, 4.0 µl 10x reaction buffer,
4.0 µl 75 mM ATP, 4.0µl 75 mM GTP, 3.0 µl 75 mM CTP, 3.0 µl 75 mM UTP, 7.5 µl 10 mM
Biotin 11-CTP, 7.5 µl 10 mM Biotin 11-UTP (PerkinEliner Life Sciences Inc. Boston,
MA, USA) and 4.0 µl enzyme mix. The reaction mix was incubated for 14 h at 37°C and
cRNA target purified using an RNeasy
® kit (Qiagen). cRNA yield was quantified by measuring the UV absorbance at 260 nm,
and fragmented in 40 mM Tris-acetate (TrisOAc) pH 7.9, 100 mM KOAc and 31.5 mM MgOAc,
at 94°C for 20 min. This results typically in a fragmented target with a size range
between 100 and 200 bases.
Array, hybridization
[0249] Ten micrograms of fragmented target cRNA was used for hybridization of each UniSet
Human I Expression Bioarray chip (AmershamBiosciences), in 260 µl of hybridization
solution containing 78 µl Amersham Hyb buffer component A and 130 µl Amersham Hyb
buffer component B. The hybridization solution was heated at 90°C for 5 min to denature
the cRNA and chilled on ice. The sample was vortexed for 5 s at maximum speed, and
250 µl injected into the inlet port of the hybridization chamber. The slides were
loaded into a ISF-4-W shaking incubator (Kuhner, Birsfelden, Switzerland), with the
hybridization chambers facing up. Slides were incubated for 24 h at 37°C, while shaking
at 300 r.p.m.
Post-hybridization processing, using streptavidin-Cy5
[0250] The slides were removed from the ISF-4-W shaker, and the hybridization chamber removed
from each slide. Each slide was briefly rinsed in TNT buffer (0.1 M Tris-HCl pH 7.6,
0.15 M NaCl, 0.05% Tween-20) at room temperature, and then washed in TNT buffer at
42°C for 60 min. The signal was developed using a 1:500 dilution of streptavidin-Cy5
(AmershamBiosciences), for 30 min at room temperature. Excess dye was removed by washing
four times with TNT buffer, for 5 min each, at room temperature. Slides were rinsed
in 0.05% Tween-20 and dried under nitrogen gas. Processed slides were scanned using
an Axon GenePix 4000B Scanner with the laser set to 635 nm, the photomultiplier tube
(PMT) voltage to 600 and the scan resolution to 10 µm. Images were acquired with the
Axon GenePixPro v4.0 Scanning Software (AmershamBiosciences), and analyzed using the
CodeLink
™ Expression Analysis Software (AmershamBiosciences).
Data analysis
[0251] CodeLink
™ Expression Analysis Software(AmershamBiosciences) automatically creates signal data
for each spotted dot as a Microsoft Excel formatted spreadsheet. The data was then
compared using the computer program Spotfire Decision Site 7.0 (Spotfire Japan K.K.,
Tokyo, Japan) to determine the fold difference between each gene in normal and COPD
lung. As a result of this analysis, the RC Kinase gene transcript was found to be
expressed higher in COPD than in normal lung (Fig. 1, 2).
EXAMPLE 7
Tissue-specific expression of RC Kinase
[0252] The qualitative expression pattern of RC Kinase in various tissues is determined
by Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
[0253] To demonstrate that RC Kinase is involved in the disease process of COPD, 25 µg of
total RNA from the following sources were used as template in reactions to synthesize
first-strand cDNA for expression profiling : Human Total RNA Panel I-V (Clontech Laboratories,
Palo Alto, CA, USA), normal human lung primary cell lines (BioWhittaker Clonetics,
Walkersville, MD, USA), human umbilical vein endothelial cells (HUVECs) (Kurabo, Osaka,
Japan), several common cell lines (ATCC, Washington, DC), and various cells purified
from peripheral blood. First-strand cDNA was synthesized using oligo (dT) (Nippon
Gene Research Laboratories, Sendai, Japan) and the SUPERSCRIPT™ First-Strand Synthesis
System for RT-PCR (Life Technologies, Rockville, MD) according to the manufacturer's
protocol. For these samples, 1/1250
th of the synthesized first-strand cDNA was subsequently used as template for quantitative
PCR. Additional samples were purchased as presynthesized cDNAs (Human Immune System
MTC Panel and Human Blood Fractions MTC Panel, Clontech Laboratories), and for these,
10 ng of cDNA was used as template for quantitative PCR.
[0254] Quantitative PCR was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis,
IN) with oligonucleotide primers 5'-AATGGCACCCACAGTGACATGCTT-3' (SEQ ID NO: 13) and
5'-CCCTCGGTGTGCTCCGATGTAAAA-3' (SEQ ID NO: 14) in the presence of the DNA-binding
fluorescent dye SYBR Green I. Results were then converted into copy numbers of the
gene transcript per ng of template cDNA by fitting to a standard curve. The standard
curve was derived by simultaneously performing the quantitative PCR reaction on PCR
products of known concentrations amplified beforehand from the target gene.
[0255] To correct for differences in mRNA transcription levels per cell in the various tissue
types, a normalization procedure was performed using similarly calculated expression
levels of five different housekeeping genes: glyceraldehyde-3-phosphatase (GAPDH),
hypoxanthine guanine phophoribosyl transferase (HPRT), beta-actin, porphobilinogen
deaminase (PBGD), and beta-2-microglobulin. The level of housekeeping gene expression
is considered to be relatively constant for all tissues (Adams et al., 1993, Adams
et al., 1995, Liew et al., 1994) and therefore can be used as a gauge to approximate
relative numbers of cells per ng of cDNA template. Expression levels of the five housekeeping
genes in all tissue samples were measured in three independent reactions per gene
using the LightCycler and a constant amount (25 µg) of starting RNA. The calculated
copy numbers for each gene, derived from comparison with simultaneously reacted standards
of known concentrations, were recorded and converted into a percentage of the sum
of the copy numbers of the gene in all tissue samples. For each tissue sample, the
sum of the percentage values for each gene was calculated, and a normalization factor
was calculated by dividing the sum percentage value for each tissue by the sum percentage
value of one of the tissues arbitrarily selected as a standard. To normalize an experimentally
obtained value for the expression of a particular gene in a tissue sample, the obtained
value was multiplied by the normalization factor for the tissue tested. This normalization
method was used for all tissues except those derived from the Human Blood Fractions
MTC Panel, which were normalized against the single housekeeping gene, beta-2-microglobulin,
due to wide variation in other housekeeping gene expression in these tissues depending
on activation status. The results of this expression profiling are given in Figure
4 and Figure 5, showing the normalized values for the copy numbers of mRNA per 10
ng of first-strand cDNA in each sample tested.
[0256] To measure the relative copy numbers of the genes in patient samples and healthy
lung samples, total RNA was prepared from frozen lung tissue obtained from four normal
individuals and two individuals diagnosed with chronic obstructive pulmonary disease
(Analytical Biological Services Inc. Wilmington, DE, USA) using Trizol
™ (Invitrogen Corp., Carlsbad, CA, USA). cDNA was then synthesized as above and used
as template for quantitative PCR as described above. Normalization was performed using
the single housekeeping gene GAPDH. The different levels of expression of the RC Kinase
gene transcript between the normal and COPD lung tissue samples are shown in Figure
3, which displays the ratio of RC Kinase transcript to GAPDH transcript measured in
each sample tested.
EXAMPLE 8
[0257] Cloning of the RCKinase cDNA Due to discrepancies between the nucleotide sequence
of RC Kinase available in public databases (GenBank accession number NM_025052, length
2022 bp), and ESTs matching to this gene locus, an attempt was made to obtain the
correct full-length sequence using RACE (rapid amplification of cDNA ends). RACE templates
for this purpose were synthesized from human lung tissue using the GeneRacer
™ kit (Invitrogen Corp., Carlsbad, CA, USA). PCR amplification of the RC Kinase cDNA
was then carried out using the gene-specific oligonucleotide primer 5'-CCCTCGGTGTGCTCCGATGTAAAA
-3' (SEQ ID NO: 14) and the GeneRacer 5' primer included in the kit. The PCR amplification
generated a product of approximately 5 kilobases in length, indicating a significantly
longer transcript than that registered in the GenBank database.
[0258] To amplify the full-length sequence of RC Kinase, PCR primers were designed within
predicted upstream exons and downstream exons of the RC Kinase gene. The upstream
exons were predicted by aligning the mouse ortholog of RC Kinase, generated by the
gene prediction program GenomeScan and registered in GenBank with the accession number
XM_136210, with the genomic locus of human RC Kinase. Regions of alignment were considered
to be potential exons of the human gene. Downstream exons were predicted by alignment
of ESTs with the genomic locus and selecting the 3'-most regions of alignment. Primers
used for amplification of the full-length sequence of RC Kinase were 5'-TTCAAAGAAACAGCAGCTTTTGGACATT-3'
(SEQ ID NO: 15) and 5'-GCATCTGCAGTGGAACTGGGAAGAA -3' (SEQ ID NO: 16).
[0259] PCR products were cloned into a pCR4-TOPO sequencing vector (Invitrogen, Carlsbad,
CA), cycle-sequenced with an ABI Prism Dye Terminator Cycle Sequencing Reaction Kit
(Applied Biosystems, Foster City, CA), and analyzed on an ABI Prism 377 sequencing
system (Applied Biosystems).
EXAMPLE 9
[0260] Because the occurrence of COPD is strongly correlated with the long-term repeated
inhalation of irritants contained in tobacco smoke, the applicants performed an analysis
to determine the influence of various cellular stress inducing compounds on the expression
of RC Kinase. As shown in figures 6 and 7, the expression of RC Kinase in the cell
lines HEK293, Jurkat, and Daudi increased significantly after treatment of the cells
with 95 mM potassium chloride (KCl), which subjects the cells to a hyperosmotic stress.
Additionally, the Jurkat cell line (and to a smaller degree, the Daudi cell line)
increased its expression of RC Kinase in response to 500 µm hydrogen peroxide (H
2O
2), a treatment which subjects the cells to an oxidative stress and which has been
reported to impair the capacity of B cells to stimulate specific T cells. Such upregulation
of RC Kinase in the cell lines in response to hyperosmotic and oxidative stress suggests
that higher expression of RC Kinase in the lungs of COPD patients may be the result
of cellular stresses caused by the irritants in tobacco smoke or stresses caused by
the inflammatory response to those irritants.
EXAMPLE 10
[0261] In order to determine whether the overexpression of RC Kinase has the potential to
activate transcription factors, the applicants used a luciferase reporter gene assay
to measure specific transcription factor activation. As shown in figure 9, the transfection
and overexpression of RC Kinase lead to the activation of the transcription factors
AP-1 and NFκB. Very little activation of the transcription factor NFAT could be detected.
The consequences of this transcription factor activation were then studied by the
applicants using microarrays to analyze which genes show increased transcription when
RC Kinase is overexpressed. As a result, it was found that the expression of the chemokine
Interleukin-8 was strongly increased in RC Kinase transfected cells. To confirm this
result, the amounts of Interleukin-8 in the culture medium from cells transfected
with an RC Kinase expression vector and from cells transfected with an empty vector
were compared by enzyme-linked immunosorbent assay. As shown in figure 10, overexpression
of RC Kinase causes a nearly 35-fold increase in the production of Interleukin-8 by
the cells. Therefore, as illustrated in figure 11, RC Kinase appears to be upregulated
and possibly activated by cellular stress, then phosphorylates MKK4 (and to a lesser
extent MKK6), which leads to the activation of transcription factors AP-1 and NFκB.
As a result of activation of this signaling cascade, Interleukin-8 production is increased
and leads to the recruitment of inflammatory cells, such as neutrophils, that play
a role in the pathology of COPD.
EXAMPLE 11
Measurement of Interleukin-8 production by cells expressing RC Kinase
[0262] Levels of h-IL-8 in the culture media of HEK293 cells expressing RC Kinase were measured
using MAXISORP 96-well flat-bottom plates (Nunc, Roskilde, Denmark) using the Duoset
ELISA Development System for human IL-8 (Genzyme Diagnostics, Cambridge, MA).
[0263] Culture medium was collected from HEK293 cells two days after transfection with an
empty vector or an RC Kinase expression vector. 100 µl of the collected medium was
diluted and put into wells of plates that had been precoated overnight with primary
antibody and blocked with 300 µl of 1% BSA-PBS for 1h at room temperature. The plates
were then incubates for 2 h at room temperature. After washing the plates twice with
300 µl of PBS-0.05% Tween, 100 µl of 0.25 µg/ml secondary antibody diluted with PBS-0.1%
BSA-0.05% Tween was added to the wells and the plates were incubated for an additional
1 h at room temperature. The plates were then washed three times with 300 µl of PBS-0.05%
Tween. 100 µl of 0.25 µg/ml detection antibody (streptavidin-HRP) diluted with PBS-0.1%
BSA-0.05% Tween was added, and the plates incubated for 1 h at room temperature. After
washing the plates four times with 300 µl of PBS-0.05% Tween, the plates were developed
using a peroxidase mediated color (Sumitomo Bakelite, Tokyo, Japan) and the absorbance
was measured at OD 450 nm. The concentration of Interleukin-8 was then calculated
by comparing the measured absorbance against the absorbance of serially diluted IL-8
standards prepared simultaneously.
[0264] As a result, it was shown that overexpression of RC Kinase in HEK293 cells causes
an approimate 35 fold increase in the production of Interleukin 8 (Fig. 10).
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